U.S. patent application number 09/807723 was filed with the patent office on 2003-02-27 for mutiple gene expression for engineering novel pathways and hyperexpression of foreign proteins in plants.
Invention is credited to Daniell, Henry, Moar, William.
Application Number | 20030041353 09/807723 |
Document ID | / |
Family ID | 25197056 |
Filed Date | 2003-02-27 |
United States Patent
Application |
20030041353 |
Kind Code |
A1 |
Daniell, Henry ; et
al. |
February 27, 2003 |
Mutiple gene expression for engineering novel pathways and
hyperexpression of foreign proteins in plants
Abstract
Introducing blocks of foreign genes in a single operon would
avoid complications such as position effect and gene silencing
inherent in putting one gene at a time into random locations in the
nuclear genome. Cloning several genes into a single T-DNA does not
avoid the compounded variable expression problem encountered in
nuclear transgenic plants. This disclosure shows that a bacterial
operon can be expressed in a single integration event as opposed to
multiple events requiring several years to accomplish. Expression
of multiple genes via a single transformation event opens the
possibility of expressing foreign pathways or pharmaceutical
proteins involving multiple genes. Expressing the Cry2aA2 operon,
including a putative chaperonin to aid in protein folding, in the
chloroplast via a single transformation event leads to production
of crystalized insecticidal proteins. Expressing the Mer operon via
a single transformation event leads to a phytoremediation
system.
Inventors: |
Daniell, Henry; (Winter
Park, FL) ; Moar, William; (Auburn University,
AL) |
Correspondence
Address: |
Schnader Harrison Segal & Lewis
IP Department 36th Floor
1600 Market Street
Philadelphia
PA
19103
US
|
Family ID: |
25197056 |
Appl. No.: |
09/807723 |
Filed: |
April 18, 2001 |
PCT Filed: |
February 28, 2001 |
PCT NO: |
PCT/US01/06276 |
Current U.S.
Class: |
800/288 |
Current CPC
Class: |
C12N 15/8214 20130101;
C12N 15/8286 20130101; Y02A 40/146 20180101; C12N 15/8259 20130101;
C12N 15/79 20130101; C12N 15/8241 20130101; C12N 15/8257 20130101;
C07K 14/325 20130101; Y02A 40/162 20180101; C12N 15/74 20130101;
C12N 15/8216 20130101; C12N 15/8271 20130101 |
Class at
Publication: |
800/288 |
International
Class: |
A01H 005/00 |
Goverment Interests
[0002] The work of this invention is supported in part by the
USDA-NRICGP grants 95-82770, 97-35504 and 98-0185 to Henry Daniell.
Claims
1. A stable chloroplast transformation and expression vector which
is capable of introducing multiple genes into a selected plant by a
single integration event, wherein each step of said multiple genes
is carried out by an enzyme encoding a heterologous DNA sequence
which comprises an expression cassette, comprising as operably
linked components, in the 5' to the 3' direction of translation, a
promoter operative in said plastids which drives a multi-gene
operon, a selectable marker sequence, the multi-gene operon which
is functional to co-express multiple enzymes in the plastids, a
transcription termination region functional in said plastids, and
flanking each side of the expression cassette, flanking DNA
sequences which are homomlogous to DNA sequences inclusive of a
spacer sequence off the target plastid genome, whereby stable
integration of the heterologous coding sequence into the
chloroplast genome of the target plant is facilitated throughout
homologous recombination of the flanking sequence with the
homologous sequences in the target plastid gene.
2. A vector of claim 1, wherein a gene of the operon codes for an
insecticidal toxin crystal protein.
3. A vector. of claim 2, wherein the insecticidal toxin crystal
protein is a Bacillus thumngensis (Bt) crystal protein.
4. A vector of claim 3, wherein another gene of the operon codes
for another insecticidal crystal protein with a different mode of
action
5. A vector of claim 4, wherein the multi-gene operon is functional
to co-express, in addition to a Bt insecticidal toxin gene, a
non-Bt insecticidal toxin gene selected from at least one of the
group of cholesterol oxidase, alpha-amylate inhibitors, protease
inhibitors, cowpea trypsin inhibitors and the potato proteinase
inhibitor II, whereby the "pyramiding" of the toxin product tends
to retard the ability of insects to adapt to the insecticidal
effect of the transgenic target plants.
6. A vector of claim 4 or 5, wherein a second gene of the operon
codes for a putative chaperonin which facilitates the folding of
the Bt crystal toxin protein to form proteotically stable cuboidal
crystals.
7. A vector of claim 3, wherein the operon includes one of the 133
genes shown in the article MMBR, (September, 1998, pages 805-873,
Vol 62, No. 4, Revision of the Nomenclature for the Br-Pesticidal
(insecticidal) Crystal Proteins by Crickmon, et al.), wherein at
least one of the genes of the operon codes for a Bt insecticidal
crystal protein and another gene codes for a putative corresponding
chaperonin which facilitates the folding and the corresponding
chaperonin facilitates the folding of the Bt protein to
proteolytically stable cuboidal crystals shown in that MMBR
article.
8. A vector of claim 1, wherein at least one of the gene of the
operon codes for a biopharmaceutical protein.
9. A vector of claim 7, wherein another gene of the operon codes
for a putative chaperonin which facilitates the folding of the
protein.
10. A vector of claim 8, wherein the protein is insulin or human
albumin.
11. A vector of claim 7, wherein another gene of the operon codes
for another biopharmaceutical protein other than the gene which
codes for the putative chaperonin, which protein is expressed in
stoichimetric ratio.
12. A vector of claim 10, wherein the genes of the operon, other
than the gene which codes for the putative chaperonin, codes for
biopharmaceutical proteins which are expressed in stoichimetric
ratio.
13. A vector of claims 7,8,9 or 10 which comprises collecting the
protein product in a folded configuration, thereby enhancing their
stability, and facilitating single step purification.
14. A method of combating insects which comprises applying to the
insects or their habitat an insecticidally amount of the
insecticidal crystal protein of claim 4.
15. A method of transforming a chloroplast of a selected plant
species or the progeny thereof to confer insect resistance and
producing on a large-scale foreign protein, said method comprising
the steps of: stably transforming the chloroplast of selected plant
cells to express at least one insecticidal toxin protein and a
chaperonin, growing the transformed plant cells under conditions
which allow the expression of said insecticidal toxin protein and
chaperonin.
16. The method of claim 14, further comprising the steps of
culturing said plant cells in a plant growth medium comprising
spectinomycin, and selecting transformed plant cells capable of
growth in the presence of said spectinomycin.
17. The method of claim 15, further comprising regenerating a
transformed plant from said transformed plant cells.
18. A transformed plant which has been transformed by the method of
any one of claims 14-16.
19. The transformed plant of claim 18, wherein said plant contains
a high accumulation of insecticidal toxin proteins in said plant's
leaves, including mature and old bleached leaves.
20. The progeny, including seeds, of the transformed plant of claim
18.
21. A vector of claim 1, wherein the biosynthetic pathway is a
bioremediation system that functions to degrade inorganic and
organic metal compounds in contaminated sites.
22. A vector of claim 21, wherein the expression cassette does not
contain a terminator.
23. A vector of claim 21 or claim 22, wherein the operon contains
the merry resistance coding sequences encoding enzymes Mer A and
Mer B.
24. The vector of claim 23, wherein the bioremediation pathway is
driven by a single promoter.
25. The chloroplast transformation and expression vector of claim
24, wherein enzymes of the bioremediation pathway are expressed in
stoichiometric amounts.
26. A vector of claim 25, wherein the inorganic compounds are
selected from at least one of the group consisting of divalent
cations of mercury, nickel, cobalt, trivalent cations of gold, and
monovalent cations of silver.
27. A vector of claim 25, wherein the organic compounds are
selected from at least one of the group consisting of alkyl
mercury, alkenyl mercury, alkynyl mercury, aromatic mercury
compounds, alkyl lead compounds, alkyl arsenic compounds and alkyl
cadmium compounds.
28. A method of transforming a chloroplast of a selected plant
species or the progeny thereof to confer greater resistance to
metal ions than the corresponding parental plant which does not
require several back crosses to create complete pathway that
detoxifies mercury and organiomercurial, said method comprising the
steps of: stably transforming the chloroplast of a plant by
inserting an expression cassette containing the mercury resistance
coding sequences of claim 21 into a plant species or the progeny
thereof, growing the transforming plant species under conditions
which allow the expression of said expression cassette.
29. The method of claim 28, further comprising culturing said plant
in a plant growth medium comprising a selector for the
corresponding selectable marker of claim 1, and selecting
transformed plant cells capable of growth in the presence of said
selector.
30. The method of claim 29, further comprising regenerating a
transformed plant from said transformed plant cells.
31. A stably transformed plant which has been transformed by the
methods of any one of claims 28-30.
32. The progeny, including seeds, of the stably transformed plant
of claim 31.
33. A method of phytoremediation of mercury and organomercurials in
soil and ground water, said method comprising the steps of:
planting the stably transformed plant of either claim 31 or claim
32 in soil contaminated with mercury and organomercurials and
allowing said plants to grow.
34. A method of phytoremediation which does not require several
back crosses to create complete pathway that detoxifies mercury and
organomercurial, said method comprising the methods of claims
28-30.
35. The plants of claim 33, wherein the plant contains, products of
the bioremediation pathway.
36. The products of the stably transformed plant of either claim 31
or claim 32, wherein said products are metals that are reduced by
the enzymes of the bioremediation pathway.
37. The vector of claim 23 which is capable of introducing a
multiple-step biosynthetic pathway into a selected photosynthetic
cell by a single integration event
38. The vector of claim 37, wherein the biosynthetic pathway
degrades inorganic and organic mercury compounds.
39. A vector of claim 38, wherein the bioremediation pathway is
driven by a single promoter.
40. A vector of claim 38, wherein the enzymes of the bioremediation
pathway are expressed in stoichiometric amounts.
41. A vector of claim 38, wherein the inorganic compounds are
selected from at least one of a group consisting of divalent
cations of mercury, nickel, cobalt, trivalent cations of gold, and
monovalent cations of silver.
42. A vector of claim 38, wherein the organic compounds are
selected from at least one of a group consisting of alkyl mercury,
alkenyl mercury, alkynyl mercury, aromatic mercury compounds, alkyl
lead compounds, alkyl arsenic compounds and alkyl cadmium
compounds.
43. A photosynthetic organism transformed with the vector of claim
38 which is useful for bioremediation of mercury and
organomercurial compounds from contaminated water bodies.
44. A method of transforming a chloroplast of a selected
photosynthetic organism to confer greater resistance to metal ions,
said method comprising the steps of: stably transforming the
chloroplast of a photosynthetic organism with the vector of claim
38, growing the transformed photosynthetic organism under
conditions which allow the expression of said expression
cassette.
45. The method of claim 44, further comprising culturing said
photosynthetic organism in a growth medium comprising a selector,
and selecting transformed cells capable of growth in the presence
of said selector.
46. The method of claim 45, further comprising regenerating a
transgenic photosynthetic organism from said transformed cells.
47. A method of phytoremediation of mercury and organomercurials in
bodies of contaminated water, said method comprising the steps of:
treating water contaminated with mercury and organomercurials with
the transgenic photosynthetic organism of claim 42 before releasing
the water into the environment.
48. The photosynthetic organism of claim 43, wherein said
photosynthetic organism is either a green algae or a
cyanobacteria.
49. The photosynthetic organism of claim 48, wherein the green
algae is Chlorella vulgaris.
50. The photosynthetic organism of claim 48, wherein the
cyanobacteria is Synechocytis.
51. A vector of claim 1 wherein a multi-gene operon codes for a
protein.
52. A vector of claim 51, wherein the protein is a
biopharmaceutical protein.
53. A vector of claim 52, wherein the biopharmaceutical protein is
a monoclonal antibody.
54. A vector of claim 53, wherein the protein is produced in the
same stoichiometric ratio.
55. A vector of claim 4, wherein said another gene of the operon is
selected from the group of cholesterol oxidase, alpha-amylase
inhibitors, and proteinase inhibitors.
56. The vector of anyone of claims 1-13, 21-27, 37-42, 51-55,
wherein the promoter is a one functional in green or non-green
plastids.
57. The promoter of claim 56, wherein said promoter is selected
from the group of psbA, accD, or 16srRNA promoters.
58. The biosynthetic pathway of claim 1, wherein said biosynthetic
pathway result in the production of compounds such as amino acids,
fatty acids, carbohydrates, polymers, vitamins, antibiotics and
dyes.
59. A vector of claim 8, where the protein is human serum
albumin.
60. A vector of claim 1, which further comprises flanking each side
of the expression cassette, flanking DNA sequences which are
homologous to a DNA sequence inclusive of a spacer sequence of the
target chloroplast genome, which sequence is conserved in the
chloroplast genome of different plant species, whereby stable
integration of the heterologous coding sequence into the
chloroplast genome of the target plant is facilitated through
homologous recombination of the flanking sequences with the
homologous sequences in the target chloroplast genome.
Description
RELATED APPLICATIONS
[0001] This patent application claims the benefit of U.S.
Provisional Applications Nos. 60/185,660, filed Feb. 29, 2000,
60/257,408, filed Dec. 22, 2000, 60/259,248 filed Dec. 29, 2000 and
60/266,121 filed Feb. 2, 2001. All applications are here
incorporated by reference.
FIELD OF THE INVENTION
[0003] This application pertains to the field of genetic
engineering of plant genomes, particularly plastids and to methods
of and engineered plants with operons that lead to and result in
overexpression of the gene of interest. This application also
pertains to the field of genetic engineering of algal and bacterial
genomes.
DESCRIPTION OF RELATED ART
[0004] Karamata, in U.S. Pat. No. 4,797,279, proposed the
generation of Bacillus thuringiensis hybrids that have insecticidal
properties through conjugation. Conjugation is mediated by a
conjugative plasmid functional in the B.t. kurstaki strain and the
B.t. tenebrionis strain. The resulting hybrid is capable of
producing each of the delta-endotoxin crystals typical for a B.t.
kurstaki strain and a B.t. tenebrionis strain.
[0005] McBride, in U.S. Pat. No. 5,545,818 and McBride et al.
(1995), describes a method of genetically engineering the plastids
of a plant or plant cell such they provide increased expression of
the Bacillus thuringiensis insecticidal proteins in the plastids. A
construct containing a promoter functional in plant plastids, a
single gene encoding an insecticidal Bacillus thuringiensis toxin,
another DNA sequence encoding a selectable marker, and a
transcription termination region capable of terminating
transcription in a plant plastid, is used to affect plant
transformation. The transcription and translation of the B.t gene
product occurs in the plastids.
[0006] Daniell et. al., in U.S. Pat. No. 5,932,479 (1999), entitled
"Genetic engineering of plant chloroplast," teaches plant cells
chloroplast transformed by means of an expression cassette
comprising an exogenous DNA sequence which is stably integrated to
the chloroplast genome of the cell of a target plant. "Stably"
integrated DNA sequences are those which are inherited through
genome replication by daughter cells or organisms. This stablility
is exhibited by the ability to establish permanent cell lines,
clones, or transgenic plants comprised of a population containing
the exogenous DNA.
[0007] Likewise, U.S. Pat. No. 5,693,507 (1997) to Daniell and
McFadden discloses such stable integration of the chloroplast by
means of an expression cassette which comprises an exogenous DNA
sequence which codes for a desired trait, and the transformed
plant.
[0008] Daniell, in PCT International Publication WO 99/10513,
teaches the composition and use of universal chloroplast
integration and expression by vectors to stably transform and
integrate genes of interest into chloroplast genome of multiple
species of plants. This leads to chloroplast expression of genes of
interest. Transformed plants show the highest level of expression.
Plants transformed with insecticidal genes are lethal to insects
that are 40,000-fold resistant to Bt. insecticidal proteins.
[0009] Significantly, in the prior art inventions use multiple
promoters to drive the expression of multiple genes. Put
differently, the inventions of the prior art employ a single
promoter to drive a single monocistron. In contrast, the present
invention employs a single promoter to drive polycistrons,
resulting in equal levels of expression of the polycistrons.
[0010] All publications and patents are hereby incorporated by
reference.
BACKGROUND OF THE INVENTION
[0011] In plant and animal cells, nuclear mRNAs are translated
monocistronically. This poses a serious problem when engineering
multiple genes in plants. Therefore, in order to express the
polyhydroxybutyrate polymer or Guy's 13 antibody, single genes were
first introduced into individual transgenic plants, then these
plants were back-crossed to reconstitute the entire pathway or the
complete protein. Similarly, in a seven year-long effort, Ye et al.
recently introduced a set of three genes for a short biosynthetic
pathway that resulted in .beta.-carotene expression in rice. ID
contrast, most chloroplast genes of higher plants are
co-tanscribed. Multiple steps of chloroplast mRNA processing are
involved in the formation of mature mRNAs.
[0012] In accordance of the invention expression of polycistrons
via the plastid genome, in particular the chloroplast genome,
provides a unique opportunity to express entire pathways in a
single transformation event. Additionally, chloroplast genetic
engineering is an environmentally friendly approach resulting in
containment of foreign genes and hyper-expression.
[0013] Plant bioremediation (phytoremediation), is the use of
plants for in-situ restoration of contaminated sites. The technique
has risen in the last decade as a strong and safe technique to
address with the increasing problems of the pollution of soil and
water bodies. In the past other techniques, such as mechanical and
bacterial bioremediation were implemented with little success,
since they were costly and threatened the safety of our
environment. Plants, on the other hand, are advantageous for
bioremediation systems since they have a high capacity for
adaptation to different environments and a natural resistance
against different toxic pollutants. They are cheap, non-evasive and
help contain disrupted ecosystems. These characteristics make
plants an ideal vehicle for bioremediation.
[0014] Mercury is a toxic heavy metal that is commonly released
into the environment as a byproduct of different chemical reactions
of modern industries. The present world production of mercury is
about 9000 tons/year (http://www.chem.ualberta.cal.htm). In the
environment, mercury is rapidly methylated by methanogenic bacteria
(Ex Desulfovibrio desulfuricans) producing the 10 fold more toxic
organomercurials (Compeau et. al.) 1985; Gilmour et al. 1992).
Organomercurials are more toxic due to its increased
hydrophobicity, which allows it to cross lipid membranes because it
is more hydrophobic than mercury. Over 90% of the intake of
methylmercury is absorbed into blood compared with only 2% of
inorganic mercury (http://www.chem.ualberta.cal.htm). Both
organomercurials and mercury have the tendency to accumulate in the
tissue, especially in the membrane bound organelles. In plants
organic mercury crosses the lipid membrane of organelles, for
example chloroplast, where it can poison essential oxidative and
photosynthetic electron transport chains more easily than metallic
mercury (Rugh et. al. 1996). In photosynthetic organisms, mercury
affects the oxygen-evolving complex that is found in the
photosystem II and is bound to the thylakoid membrane (Bernier et
al. 1993). Mercury treatment of PSII leads to a strong inhibition
of oxygen evolution by removal of EP33 (one of the proteins of the
OEE complex; Bernier et al. 1995). Mercury reduces the Fm and Fv
values due to additional inhibitory sites on the donor side of
PSII, including damage to the light-photochemistry (Rashid et al.
1990). Medical researchers discovered that high levels of
methylmercury cause severe neurological degeneration in birds, cats
and humans (Minamata Disease Research Group, 1968; Harada et al.
1995). Thus, mercury and organomercurials are ideal targets for
phytoremediation.
[0015] In water, mercury pollution also poses a problem. Mercury
accumulates in the sediments of lakes and oceans where methanogenic
bacteria live (http://ehpnet.niehs.nih.gov). These bacteria
methylate mercury to produce methylmercury, which is eventually
released into water (Harada et al. 1995). The methylmercury is
trapped into the small fish when the water passes through their
gills or they feed on phytoplanktons that carry high concentrations
of the pollutant. Predatory fish, as bass in fresh water and tuna
in salt water, live for long periods of time feeding on smaller
fish. During their life span, they can accumulate high levels of
methylmercury that can reach 1.0 ppm in normal water and 30 ppm in
areas of high pollution with mercury (http://ehpnet.niehs.nih.gov)
Then, humans and birds feed on contaminated fish and accumulation
in their tissue cause severe neurological damage.
[0016] Meagher and colleagues have used a nuclear modified form of
the merA and merB genes to transform plants that are resistant to
mercury and organomercurials respectively (Bizily et al. 1999; Ruga
et. al. 1996), U.S. Pat. No. 5,965,796 (1999). One of the drawbacks
of nuclear genetic engineerig is that it requires several back
crosses to create the complete pathway that detoxifies mercury and
organomercurials (Bizily et al. 2000). This results in variation in
expression levels among different transgenic lines and tolerance to
different concentrations of organomercurials, only in low levels of
tolerance (10 .mu.M) (Bizily et al. 2000). Another concern of the
use of nuclear transformed plants in-situ is the escape of the
foreign genes via pollen (Daniell 1999; Bogorad, 2000).
[0017] The present invention provides a transgenic plant
bioremediation system for soil as well as a transgenic
algae/bacteria bioremediation system for water.
Non-obviousness of Expression of Operons Via the Chloroplast
Genome
[0018] Despite the potential advantages of chloroplasts for foreign
gene expression, it was not obvious that multiple genes expressed
by a single promoter in chloroplasts would be expressed in this
organelle in a coordinated manner. Polycistrons have been observed
in chloroplasts in the past but processing RNA sequences present in
between individual transcripts, proteins or enzymes involved in
processing or cofactors necessary for processing of polycistrons
have not yet been characterized Therefore, it was not obvious to
one skilled in the art that multiple foreign gene transcripts would
be properly processed and translated when expressed from a
heterologous promoter.
[0019] Prior to this patent application there were no published
reports of expression of multiple genes in chloroplasts and there
were valid reasons to suggest that it would be problematic. Indeed,
despite several reports of foreign gene expression via the
chloroplast genome, no one ever attempted expression of a bacterial
operon via the chloroplast genome because of inadequate
understanding of processing of polycistrons within plastids. All
foreign genes engineered via the plastid genome have been driven by
individual promoters and 3' regulatory sequences. It was not known
whether 3' terminators and regulatory sequences were necessary for
individual genes of the foreign operon. It is generally believed
that the proteins or enzymes involved in processing may be under
the control of the nuclear genome. It was also believed that there
maybe several environmental factors involved in processing
polycistrons, including light.
[0020] While chloroplast ribosome binding sites have been
characterized, it was not obvious that ribosome binding sites or
untranslated regions upstream of bacterial genes would function in
plastids. Also, it was not anticipated that a chaperonin present in
a bacterial cell would function within chloroplasts and help fold
the foreign protein or interfere with folding of other chloroplast
proteins. It was certainly unanticipated that it was possible to
create cuboidal crystals within chloroplasts duplicating the
functions of a bacterium during sporulation or duplicate
bioremediation pathways within plastids. There was no certainty
that the enzymes of the pathway or proteins of the operon would be
expressed in a coordinated manner.
[0021] Indeed, the prior art suggested that there might have been
unforeseen deleterious effects of high-level expression of several
foreign proteins within chloroplasts on plant growth or development
that were not apparent from the experiences with other transgenes.
The pH and oxidation state of the chloroplast differs from that of
bacterial cells in ways that might inhibit or prevent functions of
proteins or enzymes. Because the results of this invention
contradicted those teachings of the prior art, this invention was
characterized as breakthrough in plant biotechnology and featured
on the cover of Nature Biotechnology (the most prestigious
biotechnology journal in the world) in January 2000. Scientists
around the world have written reviews subsequent to that
publication appreciating this invention. Engineering multiple genes
in transgenic plants via the nuclear genome is not only extremely
time consuming (taking several years to accomplish) but is riddled
with problems of position effect, gene silencing etc. Therefore,
this accomplishment was characterized as the holy-grail of plant
biotechnology.
SUMMARY OF THE INVENTION
[0022] By this invention, plastid expression constructs are
provided which are useful for genetic engineering of plant cells
and which provide for enhanced expression of several foreign
proteins in plastids utilizing a single transformation event. The
transformed plastid is preferrably a metabolically active plastid,
such as the chloroplasts found in green and non-green plant tissues
including leaves and other parts of the plant. This invention opens
the door to engineering novel pathways for metabolic engineering
and gene stacking, or for multi subunit complex proteins requiring
stoichiometric and coordinated expression of multiple genes. The
plastid is preferably one which is maintained at a high copy number
in the plant tissue of interest.
[0023] The present invention is applicable to all plastids of
plants. These include chromoplasts which are present in the fruits,
vegetables and flowers; amyloplasts which are present in tubers
like the potato; proplastids in roots; leucoplasts and etioplasts,
both of which are present in non-green parts of plants.
[0024] The plastid expression constructs for use in this invention
generally include a single plastid promoter region and multiple
genes of interest to be expressed in transformed plastids. The DNA
sequence of interest may contain a number of consecutive encoding
regions, to be expressed as an operon, for example where
introduction of a foreign biochemical pathway into plastids is
desired for metabolic engineering or gene stacking. Plastid
expression constructs of this invention is linked to a construct
having a DNA sequence encoding a selectable marker which can be
expressed in a plant plastid.
[0025] In a preferred embodiment, transformation vectors for
transfer of the construct into a plant cell include means for
inserting the expression and selection constructs into the plastid
genome. This preferably comprises regions of homology to the target
plastid genome which flank the constructs.
[0026] The chloroplast vector or constructs of the invention
preferably include a universal chloroplast expression vector which
is capable of importing a desired trait to a target plant species.
Such a vector is competent for stably transforming the chloroplast
genome of different plant species which comprises an expression
cassette which is described further herein. Such a vector generally
includes a plastid promoter region operative in said plant cells
chloroplast, a gene which is linked to a multi-gene operon which
includes an ORF which codes for a putative chaperonin which
facilitates the folding of the protein to form proteolytically
stable cuboidal crystals. Preferably, one or more DNA sequences of
interest to be expressed in the transformed plastids.
[0027] The invention provides also a plastid vector comprising of a
DNA construct. The DNA construct includes a 5' part of a plastid
DNA sequence inclusive of a spacer sequence; a promoter that is
operative in the plastid; at least a heterologous DNA sequence
encoding multiple peptides of interest; a gene that confers
resistance to a selectable marker; a multi-gene operon; a
transcription termination region functional in the target plant
cells; and a 3' part of the plastid DNA sequence inclusive of a
spacer sequence. The DNA construct is flanked by DNA sequences
which are homologous to the spacer sequence of the target plastid
genome. The plastid is preferably a chloroplast. The vector
preferably includes a ribosome binding site and a 5' untranslated
region (5'UTR). A promoter operative in the green and non-green
plastids is to be used in conjunction with the 5'UTR,
[0028] The invention provides a promoter that is operative in the
green and non-green plastids of the target plant cells such as the
psbA promoter, rbcL promoter, atp.beta. promoter region, accD
promoter, and the 16SrRNA promoter.
[0029] The invention provides a gene, which can be a mutant gene,
that confers resistance, such as antibiotic resistance, to a
selectable marker like the aadA gene.
[0030] The invention provides a cassette which can be modified to
include a selectable marker, a gene encoding the chaperonin and any
desired heterologous gene. Such applications will be beneficial for
the high level production in plants of other desired protein
products as well
[0031] Further, the invention preferably provides a three-gene
insecticidal Bacillus thuringiensis (Bt) operon which shows operon
expression and crystal formation via the chloroplast genome. The
operon comprises of three operably linked components which operate
in concert as a biosynthetic pathway: a distal gene which codes for
a insecticidal protein and two open reading frames (ORF). The two
ORFs code for at least one molecule of interest and at least one
chaperonin to assist in the folding of the insecticidal protein.
The molecule of interest of this operon can be a peptide, an
enzyme, a selectable marker, or a bio-pharmaceutical, including
monoclonals.
[0032] This invention also provides for other three-gene operons,
particularly insecticidal operon or the Cry2Aa2 operon. This
invention also provides for operons of the Cry or Cyt series.
[0033] An operon of this invention further provides that the second
ORF (ORF2) codes for a putative chaperonin. A chaperonin is a
molecule which facilitates the folding and assembly of proteins to
form functional proteolytically stable cuboidal crystals. The ORF2
is operably linked to a gene encoding the insecticidal protein. The
invention provides a bacterial chaperonin that is capable of
facilitating the folding and assembly of insecticidal proteins.
[0034] This invention also provides crystalized insecticidal
proteins such as .delta.-endotoxin proteins, Cry proteins such as
the Cry2Aa2 proteins, or Cyt proteins.
[0035] In accordance of the invention, the introduction blocks of
foreign genes in a single operon would avoid complications inherent
in nuclear transformation such as position effect and gene
silencing in putting one gene at a time into random locations in
the nuclear genome. Repeated use of a single promoter causes gene
silencing (De Wilte, C. et. al. 2000). Cloning several genes into a
single T-DNA does not avoid the compounded variable expression
problem encountered in nuclear transgenic plants. This invention
shows that a bacterial operon can be expressed in a single
integration event. Expression of multiple genes via a single
transformation event opens the possibility of expressing foreign
pathways or pharmaceutical proteins involving multiple genes.
[0036] The invention provides for the demonstration of expression
of a bacterial operon or polycistrons in transgenic plants and
opens the door to engineer novel pathways in plants in a single
transformation event.
[0037] The invention provides a single vector or construct (or
cassette) which encodes more than one heterologous protein product.
This embodiment of the invention provides that a heterologous DNA
fragment that is introduced into a universal vector encodes more
than one gene. In one example shown, this invention discloses, the
DNA encodes an operon of three genes and produces proteins from at
least two genes, one of those genes encode a protein and a
chaperone protein. This aspect of the invention--to co-expressing
multiple genes--is beneficial if one skilled in the art desires to
introduce a biosynthetic pathway that comprises multiple steps into
plants. For example, a three step synthesis of a desired compound
might require three different enzymes. A single transformation will
generate a recombinant plant possessing all three heterologous
enzymes which can function in concert to produce the desired
product.
[0038] Thus another embodiment of the invention relates to the
maximal production of a heterologous protein by co-expressing it
with another polypeptide that induces crystallization of said
protein. This aspect of the invention provides the yield of
heterologous gene expression is greatly enhanced if the desired
protein is in crystal form in the transformed plant. The increased
yield is because the crystal form of the proteins protected them
from cellular proteases. This is accomplished by co-expressing the
desired gene with a second gene encoding a chaperonin that directs
crystallization.
[0039] Also, formation of crystals of foreign proteins opens a
simple method of purification via centrifugation. Plants
transformed with the cry2Aa2 operon of the invention show a large
accumulation and improved persistence of the expressed insecticidal
protein(s) throughout the life of the plant. This is most likely
because of the folding of the insecticidal protein into cuboidal
crystals, there by protecting it from proteases. This is an
environmentally friendly approach because folded crystals improve
the safety of the Bt transgenic plants. In contrast to currently
marketed transgenic plants that contain soluble CRY proteins,
folded protoxin crystals will be processed only by those target
insects that have high alkaline gut environment In addition,
absence of insecticidal protein in transgenic pollen eliminates
toxicity to non-target insects via pollen. Expression of the
cry2Aa2 operon in chloroplasts provides a model system for
hyper-expression of foreign proteins in a folded configuration
enhancing their stability and facilitating single step
purification. This is the first successful demonstration of
expression of a bacterial operon in transgenic chloroplast
plants.
[0040] The invention provides a model system for large-scale
production of foreign protein within chloroplasts in the folded
configuration enhancing their stability and facilitating
single-step purification, for example, biopharmaceuticals such as
human serum albumin (HSA) and insulin.
[0041] All known methods of transformation can be used to introduce
the vectors of this invention into target plant plastids including
bombardment, PEG Treatment, Agrobacterium, microinjection, etc.
[0042] The invention provides transformed crops, like solanaceous
plants (monocotyledonous and dicotyledonous). Preferably, the
plants are edible for mammals, including humans.
[0043] The invention provides target Bt transgenic plants which are
likely to show a more stable protein expressed at high levels in
the chloroplast throughout the growing season. It should increase
toxicity of Bt transgenic plants to target insects and help
eliminate the development of Bt resistance. The invention provides
an example of the cry2Aa2 bacterial operon is expressed in tobacco
chloroplasts to test the resultant transgenic plants for increased
expression and improved persistence of the accumulated insecticidal
protein(s).
[0044] The invention provides transformed plants including leaves
which accumulated a high percent of total soluble protein (close to
50%) in mature leaves and remain stable even in old bleached
leaves.
[0045] The invention provides transformed plants which are
resistant to difficult-to-control insects, like cotton boll worm,
which were killed 100% after consuming transgenic leaves. The
invention also provides plants which contain insecticidal protein
fold into cuboidal crystals. Plants which contain protoxin
crystals, which will be processed only by target insects that have
high alkaline gut environment, which should improve safety of Bt
transgenic plants. Also, plants are free of insecticidal proteins
in transgenic pollen, which eliminates toxicity to non-target
insects via pollen such as Monarch butterfly larvae. The invention
provides electron microscopic proof of the presence of the cuboidal
crystals inside the chloroplast.
[0046] The invention also provides an environment friendly approach
to engineering insect resistance to plants because folded crystal
products improve the safety of the Bt transgenic plants which will
be edible consume, mammals, including humans.
[0047] In another embodiment of the invention provides heterologous
DNA sequences which mediate resistance to (a) heavy metal in
transgenic plants or plant cells which express these coding
sequences encoding metal ion reductases and (b) organomercurial
compounds in transgenic plants or plant cells which express the
coding sequence encoding organomercury lyase. Preferably the coding
sequence is that of merA (which encodes mercuric ion reductase) and
merB (which encodes organomercury lyase).
[0048] The present invention provides a chloroplast universal
vector which contains a Mer operon containing metal resistance
coding sequences operably linked to transcriptional and
translational control sequences which are functional in the
chloroplast in target plants. Preferably the coding sequence is
that of merA and merB. Also, the present invention provides
transgenic plant cells, plant tissue and plants whose chloroplast
has been modified to contain and express two metal resistance
coding sequences operably linked to transcriptional and
translational control sequences which are functional in the
chloroplast of target plants. Preferably the coding sequences are
that of merA and merB. Also provided by the present invention are
methods for effecting metal resistance inplants by stably
transforming a plant to contain and express two heterologous DNA
sequences encoding metal resistance operably linked to
transcriptional and translational control sequences which are
functional in the chloroplasts of target plants. Preferably the
coding sequence is that of merA and merB.
[0049] The present invention are methods for effecting metal
resistance in plants by stably transforming a green algae or
cyanobacteria to contain and express two heterologous DNA sequences
encoding metal resistance operably linked to transcriptional and
translational control sequences which are functional in the target
green algae or cyanobacteria. Preferably the coding sequence is
that of merA and merB.
[0050] A further object of the invention are transgenic plants,
algae and bacteria which contain and express metal resistance and
organomercurial compound resistance coding sequences.
[0051] Other embodiments of the invention are described in greater
detail hereinafter.
BRIEF DESCRIPTION OF THE DRAWINGS
[0052] FIG. 1 shows (a) Chloroplast expression vector pLD-BD
Cry2Aa2 operon (9.8 kb) with PCR primer landing sites and expected
fragment sizes. PCR analysis of untransformed and putative
chloroplast transformants using two primer sets: (b) 1P1M; and (c)
3P3MN Lane 1: 1 kb ladder; Lane 2: untransformed; Lanes 3-7: pLD-BD
Cry2Aa2 operon putative transformants; Lane 8: pLD-BD Cry2Aa2operon
plasmid DNA.
[0053] FIG. 2 shows the Southern blot analysis of T.sub.0 and
T.sub.1 generations. A. The 0.81 kb probe BamH1-Bglll fragment
containing the chloroplast flanking sequence. Untransformed plants
generate a 4.47 kb fragment. B. Vector map showing Cry2Aa2 operon
transformed 32P labeled fragments of 8.42 kb and 1.42 kb. C. Lane
1: 1 kb ladder; Lane 2: untransformed; Lanes 3-7; T0 transgenic
lines; Lanes 8-9: T1 transgenic lines.
[0054] FIG. 3 shows the 10% SDS-PAGE gel stained with R-250
Coomassie Blue. Loaded protein concentrations are provided in
parenthesis. Lanes 1: prestained protein standard; Lane 2:
partially purified Cry2Aa2 protein from E. Coli (5 .mu.g); Lane 3:
Single Gene derived Cry2Aa2 pellet extract solubilized in 50 mM
NaOH (22.4 .mu.g); Lane 4: Single Gene derived Cry2Aa2 supernatant
(66.5 .mu.g); Lane 5: Operon Derived Cry2Aa2 supernatant (58.6
.mu.g); Lane 7: untransformed tobacco pellet extract solubilized in
50 mM NaOH (29.8 .mu.g); Lane 8: untransformed tobacco supernatant
(30.4 .mu.g). Colored compounds observed in the supernatant of
transgenic plants interfered with the DC Bio-Rad protein assays but
not in the pellet.
[0055] FIG. 4 shows the quantification of Single Gene derived
Cry2Aa2 and Operon Derived Cry2Aa2 proteins by ELISA as a
percentage of total soluble protein in young, mature, and old
transgenic leaves. A: Single Gene derived Cry2Aa2 expression shown
as a percentage of total soluble protein. B: Operon derived Cry2Aa2
expression shown as a percentage of total soluble protein.
[0056] FIG. 5 shows the insect bioassays of untransformed tobacco
leaves (A,D,G), Single Gene derived Cry2Aa2 transformed leaves
(B,E,H) and Operon Derived Cry2Aa2 transformed leaves (C,F,I). A,
B, C: bioassays with H. virescens; D, E, F: bioassays with H. zea;
G, H, I: bioassays with S. exigua.
[0057] FIG. 6 shows the electron micrographs of Operon Derived
Cry2Aa2 leaf sections in young (A), mature (BD) and old, bleached
leaf (C). Single Gene derived Cry2Aa2 mature leaf (E), mature
untransformed leaf (F).
[0058] FIG. 7 shows the phenotypes of untransformed (A) or
transformed with the cry2Aa2 gene (B) or cry2Aa2 operon (C).
[0059] FIG. 8 shows a pLD-merAB chloroplast vector
[0060] FIG. 9 shows transformed E. Coli grown in 100 .mu.m
HgCl.sub.2. Transformed E. coli cells containing the vectors
pLD-merAB and pLD-MerAB-3'UTR grown in LB at different
concentrations of HgCl.sub.2. Plates show transformed cells growing
at 100 .mu.M HgCl.sub.2. No growth was observed in the control.
[0061] FIG. 10 shows chloroplast transgenic plants. A: Transgenic
plant shoot induction in RMOP with 500 .mu.g/ml Spec. B: Transgenic
plant root induction in MSO with 500 .mu.g/ml Spec. C: Transgenic
plant grown in soil.
[0062] FIG. 11 shows integration of the mer operon into the
chloroplast genome. A: PCR using specific primers that land in the
gene cassette (5P/2M) show a product of 3.8 kb size (clones2, 4, 5,
7, 9, 11). Clones 1 and 3 show no integration of the cassette.
Positive control, is plasmid pLD-merAB-3'UTR. Negative control is
untransformed plant DNA. B: PCR using specific primers that land
within the native chloroplast genome (3P/3M), eliminate mutants
(clone 3), showing integration of the cassette into the chloroplast
genome (clones: 1, 2, 4, 5, 6, 7,9, 11. 1.6 kb PCR product).
[0063] FIG. 12 shows the Chlorella vulgaris vector construct.
[0064] FIG. 13 shows the Synechocystis vector construct.
[0065] FIG. 14 shows the Lemna vector construct.
[0066] FIG. 15 shows the Sugarcane vector construct.
[0067] FIG. 16 shows confirmation of Lemna vector construct.
[0068] FIG. 17 shows confirmation of Sugarcane vector
construct.
[0069] FIG. 18 shows other vectors suitable for operon
expression.
DETAILED DESCRIPTION OF THE INVENTION
[0070] This invention is related to transformation of the plastid
genome applicable to all plastids of plants. These include
chromoplasts which are present in the fruits, vegetables and
flowers; amyloplasts which are present in tubers like the potato;
proplastids in roots; leucoplasts and etioplasts, both of which are
present in non-green parts of plants
[0071] The invention provides in one aspect a single vector or
construct which encodes more than one heterologous protein product.
The second aspect of the invention relates to the maximal
production of a heterologous protein by compressing it with another
polypeptide or a chaperone that induces crystallization of said
protein.
[0072] The first aspect of the invention provides that a
heterologous DNA fragment that is introduced into a plastid vector
(described below) encodes more than one gene. In one example, the
DNA encodes an operon of three genes and produces proteins from
three genes. This aspect of the invention to co-expressing multiple
genes is beneficial if one desires to introduce a biosynthetic
pathway into plants that comprises multiple steps. For example, a
three step synthesis of a desired compound might require three
different enzymes. Co-expressing all three enzymes in the
chloroplast can be accomplished according to this invention. Thus,
a single transformation will generate a recombinant plant
possessing all three heterologous enzymes which can function in
concert to produce the desired product.
[0073] The second aspect of the invention is that the yield of
heterologous gene expression is greatly enhanced if the desired
protein is in crystal form in the transformed plant, as provided by
this invention. This is accomplished by coexpressing the desired
gene with a second gene encoding a chaperone protein that directs
crystallization. Data given in the specification shows almost 100
fold greater amounts of insecticidal protein can be found in plants
co-expressing the chaperone protein versus plants having only the
gene encoding the insecticidal protein. The expression cassette
itself can be modified to include a selectable marker, a gene
encoding the chaperone protein and any desired heterologous gene.
Such applications will be beneficial for the high level production
in plants of other desired protein products as well.
[0074] A flyer aspect of the invention describes a plant
bioremediation system. A plant bioremediation system employing
chloroplast transformants have a number of advantages. First,
plants have the genetic capacity (using hundreds, even thousands,
of genes) to extract at least 16 metal cation and oxyanion
nutrients from the soil and ground water. This capacity can be
chemically and genetically manipulated to extract environmental
pollutants. Second, plants have extensive root systems to help in
this mining effort; typical estimates are as high as
100.times.10.sup.6 miles of roots per acre [Dittner, H. J. (1937)
Amer. J. Botany 24:417-420]. The root systems of various
macrophytes can reach up to 40 feet into the soil. In addition,
plants are photosynthetic and govern as much as 80% of the
available energy at any given time in most ecosystems. Through
photosystem I (a system not found in photosynthetic bacteria), they
use light energy to generate large amounts of reducing power (as
NADPH) that can be used to efficiently reduce metal ions. Plants
photosynthetically fix CO.sub.2 and reduce it to make their own
carbon/energy source. This reduced carbon energy is used by plant
roots to live heterotrophically. This redox power can also be used
to reduce toxic metal ions like Hg(II) [Rugh et al. (1996) supra].
Many plants can produce large amounts of biomass annually with the
potential both to enrich contaminated soil with carbon and
nutrients and/or remove metal ions from the soil. The site of
action of mercury within the chloroplasts, ability to express
bacterial operon via the chloroplast genome, and several other
environmental benefits of chloroplast genetic engineering make this
an advantageous system for metal remediation.
[0075] An additional benefit of the metal resistant plants is their
ability to harvest metals; precious and semi-precious metals can be
reduced and thereby trapped in plant tissues. These metals include
can include gold, silver, platinum, rhenium, copper, palladium,
nickel, zinc and cadmium, where the corresponding metal ions are
reduced by the metal resistance gene product in those plants.
[0076] In addition, this invention also introduces a novel approach
for mercury and organomercurial bioremediation in water. Two
organisms are used as model systems. One is Synechocystis, a
photosynthetic bacterium (Cyanobacterium) that grows in salt and
fresh water (in a high temperature range, from ice to hot springs).
The other is Chlorella vulgaris, a green algae that grows in fresh
water. These organisms are transformed with the merA and merB genes
(mer operon) to remove mercury and organomercurials from water.
Transformed cells could be applied for sludge treatment and in
water treatment to remove organomercury and mercury form water and
sediments before releasing them to the environment, especially from
industrial effluents that generate byproducts with mercury.
[0077] "Metal resistance" means that a non-naturally occurring
organism is not inhibited by the presence of at least one of
divalent cations of mercury, cadmium, cobalt, trivalent cations of
gold, and monovalent silver ion, at concentrations (levels) at
which a naturally occurring (wild-type) counterpart of the
non-naturally occurring organism is inhibited or exhibits symptoms
of toxicity. It is not intended that the term metal resistance
refer to resistance to unlimited concentration of metal ions, but
rather the term is relative in that it relies on comparison to the
properties of a parental strain.
[0078] A "metal resistance coding sequence" is one which encodes a
protein capable of mediating resistance to at least one metal ion,
including, but not limited to, divalent cations of mercury, nickel,
cobalt, trivalent cations of gold, and by monovalent cations of
silver. Also within the scope of this definition are mutant
sequences which determine proteins capable of mediating resistance
to divalent cations of lead, cadmium and copper.
[0079] An "organomercurial resistance coding sequence" is one whose
protein product mediates resistance to such organic mercury
compounds as alkylmercurials and certain aromatic mercurials, for
example, mono- or dimethylmercury, typically in conjunction with a
metal resistance gene such as merA. As specifically exemplified
herein, the organomercurial resistance gene is the methylmercury
lyase gene (merB) and its gene product confers resistance to
organomercurial compounds such as methymercury,
p-chloromercuribenzoate (PCMB) and p-hydroxymercuribenzoate in
conjunction with the merA gene product (mercury ion reductase).
[0080] The metal resistance protein (MerA protein, mercuric ion
reductase) is exemplified by that from Tn21, a bacterial mercury
resistance transposon originally isolated from the IncFII plasmid
NR1. In addition to reducing mercuric ions, the Tn21 MerA reduces
trivalent gold and monovalent silver cations [Summers and Sugarman
(1974) Journal of Bacteriology 119:242-249]. Monovalent silver and
certain divalent metal cations have been shown to be competitive
inhibitors of mercuric ion reduction in vitro [Rinderle et al.
(1983) Biochemistry 22:869-876]. MerA mediates resistance to
trivalent gold, divalent cobalt divalent copper and divalent nickel
cations as well as divalent ionic mercury.
[0081] It is understood that nucleic acid sequences from nucleotide
14 through nucleotide 1708, or MerApe 20, MerApe 29, MerApe 38 or
MerApe 47 will function as coding sequences synonymous with the
exemplified merApe9 coding sequence. Nucleic acid sequences are
synonymous if the amino acid sequences encoded by those nucleic
acid sequences are the same. The degeneracy of the genetic code is
well known to the art; i.e., for many amino acids, there is more
than one nucleotide triplet which serves as the codon for the amino
acid; for expression in plant cells or tissue it is desired that
codon usage reflect that of plant genes and that CpG dinucleotides
be kept low in frequency in the coding sequence. It is also well
known in the biological arts that certain amino acid substitutions
can be made in protein sequences without affecting the function of
the protein. Generally, conservative amino acid substitutions or
substitutions of similar amino acids are tolerated without
affecting protein function. Similar amino acids can be those that
are similar in size and/or charge properties, for example,
aspartate and glutamate and isoleucine and valine are both pairs of
similar amino acids. Similarity between amino acid pairs has been
assessed in the art in a number of ways. For example, Dayhoff et
al. (1978) in Atlas of Protein Sequence and Structure, Vol. 5,
Suppl. 3, pp. 345-352, which is incorporated by reference herein,
provides frequency tables for amino acid substitutions which can be
employed as a measure of amino acid similarity. Dayhoff et al.'s
frequency tables are based on comparisons of amino acid sequences
for proteins having the same function from a variety of
evolutionarily different sources.
[0082] The expression of merB in plants confers resistance to
and/or the ability to detoxify organomercurials including, but not
limited to, alkylmercury compounds wherein the alkyl group is
either straight chain or branched, alkenyl mercury compounds, allyl
mercury, alkynyl mercury compounds, aromatic mercury compounds,
wherein there are from one to about 6 aromatic rings, and other
organomercurials including but not limited to humic acid-containing
mercury compounds. The MerB protein also mediates resistance to
and/or detoxifies organo-metals including, but not limited to,
organic lead, organic cadmium and organic arsenic compounds, where
those organometals can be alkyl, aklenyl, alkynyl or aromatic metal
compounds.
[0083] Coding sequences suitable for expression in a plant are
operably linked downstream of a constitutive or a regulated
promoter construct. Transgenic plants can be constructed by use of
chloroplast universal vector containing a 5' a part of a
chloroplast spacer sequence, a promoter that is operative in the
chloroplast of the target plant cells, at least two heterologous
DNA sequences encoding merA and merB, a gene that confers
resistance to a selectable marker, a transcription termination
region functional in the target plant cells; and a 3' part of the
chloroplast spacer sequence. Alternatively, the vector may not
contain a terminator.
[0084] The mer operon-expressing plants can be used in the
remediation of mercury-contaminated soil to block the
biomagnification of methyl mercury up the food chain. Deep-rooted
trees like cottonwood and sweetgum, which inhabit bottom lands, can
be transformed to express mer A and merB. These species have roots
that grow in the same general area of the sediment as
sulfate-reducing bacteria. As the transgenic plant roots take up
methyl mercury, MerB breaks the carbon mercury bond to produce
Hg(II). Hg(II) is a highly reactive metal ion and should end up
sequestered in plant tissues bound to various thiol groups.
[0085] Hg(II) produced from the MerD reaction and additional Hg(II)
taken up from the environment through its normal mining of
nutrients is reduced to Hg(0) by the MerA reaction. Hg(0) is
released directly from the roots or transpired up the vascular
system of the plant, as are waste gasses like CO.sub.2 from some
plants [Dacey, J. W. (1980) Science 210: 1017-1019; Dacey, J. W.
(1981) Ecology 62:1137; Raven et al. (1986) In: Biology of Plants,
Worth Publishers, N.Y., p.775]. By lowering the total levels in the
soil, less methyl mercury will be produced by sulfate-reducing
bacteria. Using the MerA and MerB together in transgenic plants at
contaminated sites lowers total Hg(II) levels and destroys
environmental methyl mercury, thus preventing a large portion of
the methyl mercury from moving through the environment.
[0086] The Hg(0) entering the environment joins the enormous and
stable pool of Hg(0) in the atmosphere (Nriagu (1979) In: The
Biogeochemistry of Mercury in the Environment, (New York: Elsevier)
with half life of over one year. Because Hg(0) is not easily
returned to earth, this pool is not thought to contribute less
significantly to manmade contamination of the environment. In
contrast atmospheric Hg(II) species (i.e., mercury released from
coal burning or methyl mercury released naturally) are rapidly
returned to earth by rain and dry deposition with a half-life of
about 1-2 weeks. Thus, volatilization of relatively small amounts
of Hg(0) with good air circulation effectively removes mercury from
terrestrial and aquatic environments.
[0087] Once a transgenic plant population expressing MerA and MerB
is established, these plants efficiently process mercury. Over the
subsequent few decades these plants remove or detoxify most mercury
from at a site. Relying only on currently available biological and
chemical processing, the efflux rates of Hg(0) from mercury
contaminated sites are extremely slow. At one such government site
it is estimated that only 10 kg of the 80,000 kg present in the
soil is released as Hg(0) per year (Lindberg et al. (1995) Environ.
Sci. Tech. 29,126-135). The levels of atmospheric mercury at this
and most sites (4-10 ug/m.sup.3) are 10,000 fold below what the
EPA/OSHA recommend as the maximum allowable levels (U.S. Public
Health Service (1994) Toxicological Profile for Mercury. In:
Regulations and Advisories, U.S. Public Health Service, Washington,
D.C., pp. 261-269). Even if transgenic plants at this site
increased the efflux rate of metallic mercury 200 times, the level
of atmospheric mercury would still be 50 fold below these allowable
levels. The transgenic plants of the present invention allow the
efficient removal of toxic metal compounds such as methyl mercury
and ionic mercury from soil, sediment, and aquatic environments,
thus meeting a longfelt need for efficient bioremediation of metal
and organometal contaminated sites.
The Operons of the Vector
[0088] The cry2Aa2 Operon. The preferred embodiment of the
invention is the use of Bacillus thuringiensis (Bt) cry2Aa2 operon
as a model system to demonstrate operon expression and crystal
formation via the chloroplast genome of tobacco. This operon
contains three open reading frames (ORFs). Cry2Aa2 is the distal
gene of this operon. The ORF immediately upstream of cry2Aa2 codes
for a putative chaperonin that facilitates the folding of cry2Aa2
(and other selected target proteins) to form proteolytically stable
cuboidal crystals. Because CRY protein levels decrease in plant
tissues late in the growing season or under physiological stress, a
more stable protein expressed at high levels in the chloroplast
throughout the growing season should increase toxicity of Bt
transgenic plants to target insects and help eliminate the
development of Bt resistance. The function of the third ORF is not
yet known. The invention comprises the operon with the third gene
and also with the operon without the third gene.
[0089] The mer Operon. Another embodiment of the invention uses the
mer Operon. The genes for mercury resistance are known as Mer
genes, they are found in operons of bacterial pasmids; different
genes constitute operons, but the two most important are: the merA
that codes for the mercuric ion reductase and the merB that codes
for the organomercurial lyases (Foster, 1983; Summers et al. 1978,
1986). Mer A is a 1.7 kb gene that needs NADPH as a co-factor to
reduce mercury to a volatile, non-reactive and less toxic form of
mercury (Hg0) (Begley et al. 1986). Mer B is a 638 bp gene that
undergoes the protonolysis of organomercurials by removing the
organic group and releasing elemental mercury, which is detoxified
by mera (Jackson et al. 1982). A polycistron containing both genes
allows effective degradation of mercury and organomercurials
Alternative Operons
[0090] Other Cry or Cyt operons may be used in this invention. Any
operon which comprises at least one of the 133 genes shown in the
article MMBR, September 1998, pages 805-873, Vol 62, No. 4,
Revision of the Nomenclature for the BT Insecticidal Crystal
Proteins by Crickmon et al., the genes of which codes for the
corresponding BT protein; and the chaperonin which facilitates
protein folding can be used. Likewise, any operon which comprises
at least one of the toxins enumerated in Table 15.1 or at least one
of the of Molecular Biotechnology by Glick and Pasternak can be
used. Similarly, any operon which comprises a gene which codes for
a delta-endotoxin and the chaperonin which facilitates protein
folding can be used In addition, any operon which comprises at
least a plasmid identified in Table 13.1 of the of Molecular
Biotechnology by Glick and Pasternak can be used.
The Chaperonins
[0091] Chaperonins are a class of a protein referred to as
chaperones which has been shown to consist of helper proteins in
chain folding and assembly with the cells (Gierasch and King,
1990). They facilitate the folding and assembly of newly
synthesized polypeptide chains into fictional three-dimensional
structures by preventing off-pathway reactions during folding that
lead to aggregation (Agashe V R et. al. 2000). Chaperonins provide
a sequestered environment in which folding can proceed unimpaired
by intermolecular interactions between non-native polypeptides
(Agashe V R et. al. 2000). Those skilled in the art will be
familiar with the E. Coli chaperonins: groEL and groES (Viitanen P
V et. al. 1995), (Gierasch and King, 1990). Plant chaperonins
chaperonin-60 and chaperonin-10, which are homologous of gro-EL and
gro-ES, respectively. Homologous of the E. Coli groEL and groES
continue to be identified. For instance, a stable complex of the
chaperonins has been isolated and crystallized from the extremely
thermophilic bacterium Thermus thermophilus (Lissin N M et. al.
1992). Likewise, plant chaperonins--located both in plastids and
the cytosol, continue to be identified (Baneyx F. et. al., 1995;
Viitanen P V et. al., 1995; Burt W J et. al. 1994, Grellet F. et.
al. 1993; Bertsch U et. al., 1992). These articles are hereby
incorporated in their entirety by reference.
[0092] The preferred embodiment of this invention use of those
bacterial chaperonins that are capable of facilitating the
crystallization of the Bt endotoxin polypeptides by means of the UV
Ct vector and in the transformed plants.
The Vectors
[0093] This invention contemplates the use of vectors which are
capable of stably transforming the chloroplast genome. Such vectors
include chloroplast expression vectors such as pUC, pBlueScript,
pGEM, and al others identified by Daniell in U.S. Pat. Nos.
5,693,507 and 5,932,479. These publications and patents are herein
incorporated by reference to the same extent as if each individual
publication or patent was specifically and individually indicated
to be incorporated by reference.
[0094] Universal Vector. A preferred embodiment of this invention
utilizes a universal integration and expression vector competent
for stably transforming the chloroplast genome of different plant
species (Universal Vector). The universal vector and its
construction have been described by the earlier Publication No.
WO99/10513, International publication date: Mar. 4, 1990, which is
herein incorporated in its entirety.
[0095] The 4.0 kb cry2Aa2 operon was inserted into the universal
chloroplast expression vector pLD CtV2 (5.8 kb) to form the final
E. coli and tobacco shuttle vector pLD-BD Cry2Aa2 operon (9.8 kb)
(FIG. 1A). This vector could be used to transform chloroplast
genomes of several plant species because the flanking sequences are
highly conserved among higher plants. This vector contains the 16S
rRNA promoter (Prrn) driving the aadA gene (aminoglycoside
3'-adenylyltransferase) for spectinomycin selection and the three
genes of the cry2Aa2 operon. The terminator is the psbA 3' region
from the tobacco chloroplast genome from a gene coding for the
photosystem II reaction center component. The 16S rRNA promoter is
one of the strong chloroplast promoters recognized by both nuclear
and plastid encoded RNA polymerases in tobacco and the psbA 3'
region stabilizes the transcript of foreign genes. This construct
integrates both genes into the spacer region between the
chloroplast transfer RNA genes coding for isoleucine and alanine
within the inverted repeat (IR) region of the chloroplast genome by
homologous recombination. The integration into these transcribed
spacer regions allow the gene to be inserted without interfering
with gene coding regions. Also, each genome will contain two gene
copies due to integration into the two inverted repeat regions
resulting in a higher copy number (7,000-8,000 copies per cell) and
higher levels of expression. However, the two genes may also be
integrated outside of the IR region resulting in a lower copy
number. Chloroplast transgenic plants were obtained as described
previously by Daniell (1993, 1997).
[0096] Mer operon vectors with and without terminator: In order to
understand the role of 3'UTRs in chloroplast foreign gene
expression (in mRNA stability and transcription termination),
chloroplast vectors with and without 3' UTRs were made. The PCR
products, merA and merB were cloned independently into the pCR2.1
vector (In Vitrogen). Then, the orientation of the merB gene
integration was checked. Once the correct orientation was found,
the vector TA-merB was cut with a ClaI/EcoRV. MerA was cut with
ClaI and EcoRV, and the fragment (merA) was isolated by gel
electrophoresis. Then, the merB and merA genes were ligated. After
the ligation, both genes (merB and merA) in-frame were ready for
insertion into the pLD vector. After cutting pLD vector with PstI
to linearize the vector and to remove the terminator, the merB-merA
cassette extracted from the TA-vector was ligated into the pLD
vector (FIG. 8). The final step was to check for correct
orientation. Thus the pLD-merAB vector lacking 3' UTR was
constructed.
[0097] To make the pLD-merA.B-3'UTR, specific set of primers were
used to amplify the whole cassette from the TA-merAB vector. The 5'
primer was designed with an EcoRV site and the 3' primer with a
XbaI site. These restriction sites allowed the integration into the
pLD vector only in the right orientation.
Chloroplast Integration of Foreign Genes
[0098] Foreign gene integration into the chloroplast genome was
determined by PCR screening of chloroplast transformants (FIG.
1A,B,C). Primers were designed to eliminate spectinomycin mutants
and nuclear integration. The first primer set, 1P1M, lands one
primer (1P) on the 3' end of the 16s rRNA flanking sequence and
another primer (1M) on aadA (FIG. 1A). This is to distinguish
between spectinomycin mutants and true spectinomycin transformants.
A 1.6 kb fragment is seen in true transformants (FIG. 1B, lanes
3,5,6,7). Lane 4 shows a spectinomycin mutant with no PCR product.
Untransformed tobacco DNA (lane 2) expectedly shows no product,
while pLD-BD cry2Aa2 operon plasmid DNA in lane 8 produced the 1.6
kb fragment. The second primer set, 3P3M, lands one primer (3P) on
the native chloroplast genome adjacent to the point of integration,
and another primer (3M) on the aadA gene (FIG. 1A). This primer set
generated a 1.65 kb PCR product in chloroplast transformants (FIG.
1C, lanes 3,5,6,7). Untransformed tobacco DNA (lane 2) showed no
PCR product, and pLD-BD cry2Aa2 operon plasmid DNA in lane 8 also
showed no PCR product because 3P lands on native chloroplast DNA.
Lane 4,was negative for chloroplast integration and again proving
this transformant to be a spectinomycin mutant.
[0099] Southern blot analysis was done to further demonstrate
site-specific chloroplast integration of the 4.0 kb cry2Aa2 operon
and to determine heteroplasmy or homoplasmy (FIG. 2). Bglll
digested DNA from transformed plants produce 8.42 kb and 1.4 kb
fragments (FIG. 2B) when probed with the 0.81 kb probe (FIG. 2A)
that hybridizes to the trnI and trnA flanking sequences. Transgenic
plant DNA (T.sub.0 and T.sub.1) produced the 8.42 kb and 1.4 kb
fragments (FIG. 2C, lanes 3-9). A 4.47 kb fragment (FIG. 2A) is
seen in untransformed plant DNA (FIG. 2C lane 2). T.sub.0 plant DNA
also shows this native untransformed 4.47 kb fragment (FIG. 2C,
lanes 3-7), thereby showing heteroplasmy in the T.sub.0 generation.
This 4.47 kb native band is absent from the T.sub.1 generation
(FIG. 2C, lanes 8-9), thus indicating homoplasmy. If only a
fraction of the genomes were transformed, the gene copy number
should be less than 8,000 per cell. Confirmation of homoplasmy in
T.sub.1 transgenic lines indicates that the Cry2Aa2 operon gene
copy number could be as many as 7000-8,000 per cell.
[0100] CRY2Aa2 protein expression and quantification: Expression
profile of the operon derived (OD) Cry2Aa2 and single gene derived
(SG) Cry2Aa21 is shown on a Coomassie stained SDS-PAGE gel (FIG.
3). The primary goal of this experiment is to investigate the
location of the operon derived Cry2Aa2 protein (the pellet or
supernatant) and correlate with cuboidal crystals observed in
electron micrographs (see FIG. 6). Lane 2 contains partially
purified 65 KD Cry2Aa2 from E. coli. Because crystalline Cry2Aa2
inclusion bodies are solubilized at high alkaline pH, the 50 mM
NaOH solubilized pellet was analyzed from each plant sample after
centrifugation for 20 min at 13,000 g (lanes 3,5,7). Results show
that OD Cry2Aa2 expression forms crystalline inclusion bodies
because the protein is found mostly in the pellet after
centrifugation (lanes 5-6). In contrast, expression of SG Cry2Aa2
is observed in both the pellet and the supernatant (lanes 3-4). No
Cry2Aa2 expression was seen in untransformed tobacco in either the
supernatant or the pellet (lanes 7, 8).
[0101] Cry 2Aa2 polypeptides (FIG. 3, lanes 3, 5) were scanned
using Storm 840 Gel Scanner and Image Quant Software Molecular
Dynamics). The operon-derived expression results only in a 2.5 fold
more accumulation of Cry2Aa2 than that of single gene derived
cry2Aa2 in the pellet fraction; this does not correlate with more
than 100 fold difference observed in ELISA (FIG. 4). The reason for
this discrepancy is the extreme difference in solubilization
between SG Cry2Aa2 derived amorphous inclusion bodies and the OD
Cry2Aa2 derived cuboidal crystals, as reported previously.
[0102] Despite the large difference in protein accumulation (as
shown by ELISA and electron micrographs, FIGS. 4, 6), the
concentration of solubilized protein loaded in the pellet fraction
was similar in SG Cry2Aa2 and OD Cry2Aa2 (FIG. 3, lanes 3, 5).
Attempts to completely solubilize crystalline inclusion bodies for
SDS PAGE analysis were not successful because higher pH interfered
with gel electrophoresis and repeated dilution decreased protein
concentration below detectable levels in Coomassie stained
gels.
[0103] However, for quantification using ELISA it was possible to
completely solubilize crystalline inclusion bodies under optimal
conditions and dilute the protein to fit within the linear range of
the Cry2aA2 standard. Therefore, protein expression levels of SG
Cry2Aa2 and OD Cry2Aa2 were quantified using ELISA (FIG. 4).
Additionally, CRY protein accumulation in young, mature, and old
transgenic leaves derived from a single gene or operon was compared
to investigate their stability over time. Young, mature, and old
leaves expressed SG Cry2Aa2 at 0.014%, 0.36%, and 0.03%
respectively (FIG. 4A). Cry2Aa2 levels peaked in the mature leaf
(0.36%) and drastically declined to 0.03% as the plant senesced.
However, young, mature, and old leaves containing OD Cry2Aa2
accumulated at 34.9%, 45.3%, and 46.1% respectively (FIG. 413). As
these transgenic plants aged, OD Cry2Aa2 concentrations remained
stable and did not decline like the SG Cry2Aa2. The presence of the
operon-expressed putative chaperonin should enable the toxin to be
folded into stable crystalline structures that are protected from
degradation. Based on quantitative expression, the cry2Aa2 operon
derived expression levels are comparable to that of the RuBisCo,
the most abundant protein on earth that compromises up to 65% of
leaf soluble protein.
[0104] Insect Bioassays: Five-day-old tobacco budworm (Heliothis
virescens), ten day old cotton bollworm (Helicoverpa zea) and beet
armyworm (Spodoptera exigua) larvae consumed the entire leaf after
24 hrs on the untransformed control (FIGS. 5A, D, G). H. virescens
feeding on SG Cry2Aa2 leaves died after 5 days (FIG. 5B) while
insects died after 3 days on OD Cry2Aa2 leaves (FIG. 5C). For SG
Cry2Aa2, H. zea consumed considerable leaf material after 24 hr,
stopped feeding after three days and died after five days (FIG.
5E). H. zea consumed very little OD Cry2Aa2 material after 24
hours, stopped feeding, and died after five days (FIG. 5F). S.
exigua feeding on SG Cry2Aa2 (FIG. 5H) or OD Cry2Aa2 (FIG. 51) were
lethargic after 24 hours and died after 48 hours. Milkweed leaves
dusted with OD Cry2A2 transgenic pollen were not toxic to Monarch
butterfly larvae (data not shown) confirming earlier observations
that foreign proteins are not present in tobacco pollen.
[0105] Electron Microscopic Analysis: Untransformed and transgenic
leafsections were immunogold-labeled with a Cry2A polyclonal
antibody (FIG. 6). FIGS. A-C show developmental OD Cry2Aa2 in
chloroplasts in young, mature, and old leaves, respectively. In a
young green OD Cry2Aa2 transgenic leaf (FIG. 6A), labeled Cry2Aa2
occupies a significant amount of the chloroplast, but no
crystalline structures are observed. In a mature green OD Cry2Aa2
transgenic leaf (FIG. 6B), labeled Cry2Aa2 occupies a larger amount
of the chloroplast than the younger leaf, resulting in crystals.
Theses cuboidal crystals are essentially identical to those
expressed in wild-type Cry2Aa2 crystals, or recombinantly in Bt or
E. coli. In an old bleached OD Cry2Aa2 transgenic leaf (FIG. 6C),
labeled Cry2Aa2 maintains the crystalline structure and occupies
the highest volume of the chloroplast observed, despite being
bleached and senescent. These findings correlate with OD Cry2Aa2
ELISA results. In young developing leaves, OD Cry2Aa2 begins
accumulation (34.9%), folds Cry2Aa2 into a cuboidal configuration
in mature leaves occupying more cell volume (45.3%), and maintains
this cuboidal structure and volume in old leaves (46.1%).
Essentially, as the transgenic OD Cry2Aa2 plant ages, OD Cry2Aa2 is
accumulated, folded and maintained.
[0106] FIG. 6D is a mature green OD Cry2Aa2 transgenic leaf showing
crystal formation with no immunogold label. This probably occurs
because as the Cry2Aa2 is folded by the putative chaperonin,
epitopes are concealed thereby decreasing labeling. Crystal
formation in FIG. 6D would cause the OD Cry2Aa2 to pellet after
centrifugation as seen in SDS-PAGE FIG. 3 (lane 5). In EM analysis
of mature leaves expressing SG Cry2Aa2 (FIG. 6B), protein
aggregation is observed, although no crystalline folding is seen.
Cry2Aa2 immunogold labeling occurs in an area of much lower density
than is seen in OD Cry2Aa2 transgenic plants suggesting lower
expression. These results also correlate with ELISA (0.36% in SG
Cry2Aa2 in mature leaves). There is no localized antibody observed
in untransformed tobacco (FIG. 6F).
[0107] Transgenic phenotypes: Phenotypes of OD Cry2Aab2 transgenic
plants are not morphologically different from SG Cry2Aa2 transgenic
plants (FIG. 7). Therefore, higher levels of expression and
accumulation of CRY proteins did not visibly impact their
phenotype. Both transgenic plants flowered and set seeds.
Characterization of OD Cry2Aa2 T.sub.1 transgenic plants for stable
integration and transmission of foreign genes has been shown
earlier (FIG. 2).
Possibility of Gene Pyramiding
[0108] This invention enables expression of polycistrons in the
chloroplast genome. In contrast to prior efforts in engineering
gene expression in transgenic plants, the present invention allows
for this achievement in a single transformation event that is
environmentally safe. The invention thus opens the possibility for
gene pyramiding: the insertion of multiple insecticidal genes. The
invention contemplates operons which include not only Bacillus
thuingiensis (B.t.) insecticidal toxin genes, but also non-B.t.
insecticidal toxin genes such as cholesteral oxidase, alpha-amylase
inhibitors, protease inhibitors, the cowpea trypsin inhibitors, and
the potato proteinase inhibitor II. Inclusion of multiple
heterologous insecticidal toxin genes retards the ability of
insects to develop resistance to bio-pesticides.
Expression of Biological Pathways
[0109] Further, this invention provides a method of engineering
biological pathways into the chloroplast genome in a single
transformation event that is environmentally safe. Because gene
expression is controlled by one promoter, DNA sequences encoding
the different genes necessary in a pathway can be co-expressed to
the same levels. Once expressed, the genes of the pathway can act
in concert. Gene expression can result in synthesis of enzymes that
confer desired traits such as degradation of metal ions,
herbicides,pesticides,solvents, toulene, napthalene, and other
xenobiotics. An example is the chloroplast transformation of plant
chloroplasts with the Mer operon leading to the biodegradation of
mercury and organomercurials. Other pathways include the pigment
biosynthesis pathway, biosynthetic pathways for enzymes that are
could confer desired traits such as degradation of xenobiotic
compounds noted above, pathways for amino acids such as the lysine
biosynthetic pathway, and pathways for the synthesis of vitamins,
carbohydrates, fatty acids, biopolymers and polyesters. Further
examples are provided in chapters 12 and 13 of Molecular
Biotechnology by Glick and Pasternak, which is herein incorporated
by reference. Other xenobiotics which can be degraded using the
system of this invention include those given in U.S. Pat. No.
4,259,444 to Chakrabarty which is herein incorporated by
reference.
[0110] Expression of pathways can result in the production of
compounds such as amino acids, fatty acids, carbohydrates,
polymers, vitamins, antibiotics and dyes.
Efficient Expression of Bio-pharmaceuticals
[0111] The ability to express polycistrons also opens up the
possibility of efficiently expressing bio-pharmaceuticals such as
monoclonal antibodies. Those skilled in the art will know the four
DNA sequences encoding proteins necessary to compose the molecule.
Those skilled in the art will also know that these proteins should
be produced in equal amounts (the same stoichiometric ratio). The
PCT application entitled "Production of Antibodies in Transgenic
Plastids," filed on Feb. 28, 2001 by Daniell, is hereby
incorporated by reference to offer examples of such proteins. This
invention allows for the coordinated expression of these sequences
because they are driven by the same promoter. This method avoids
the problems of the prior art; namely the pitfalls of nuclear
transformation such as the positional effect and gene
silencing.
Application to Other Plants
[0112] This invention provides any higher plants, such as
monocotyledonous and dicotyledonous plant species. The plants that
may be transformed via the universal vector with an antibiotic
selectable marker may be solanacious plants or plants that grow
underground. Most importantly, this invention is applicable to the
major economically important crops such as maize, rice, soybean,
wheat, and cotton. A non-exclusive list of examples of higher
plants which maybe so transformed include cereals such as barley,
corn, oat, rice, and wheat; melons such as cucumber, muskmelon, and
watermelon; legumes such as bean, cowpea, pea, peanut; oil crops
such as canola and soybean; solanaceous plants such as tobacco;
tuber crops such as potato and sweet potato; and vegetables like
tomato, pepper and radish; fruits such as pear, grape, peach, plum,
banana, apple and strawberry, fiber crops like the Gossypium genus
such as cotton, flax and hemp; and other plants such as beet,
cotton, coffee, radish, commercial flowing plants, such as
carnation and roses; grasses, such as sugar cane or turfgrass;
evergreen trees such as fir, spruce, and pine, and deciduous trees,
such as maple and oak.
[0113] The invention now being generally described, it will be more
readily understood by reference to the following examples which are
included for purposes of illustration only and are not intended to
limit the present invention.
[0114] In the experimental disclosure which follows, all
temperatures are given in degrees centigrade (.degree), weight are
given in grams (g), milligram (mg) or micrograms (.mu.g),
concentrations are given as molar (M), millimolar (mM) or
micromolar (.mu.M) and all volumes are given in liters (l),
milliliters (ml) or microliters (.mu.l), unless otherwise
indicated.
[0115] The invention is exemplified in the following non-limiting
examples which are only for illustrative purposes and are not
intended to limit the scope of the invention.
EXAMPLE 1
[0116] Bombardment and selection of transgenic plants: Tobacco
plants were grown aseptically under fluorescent lights in the
laboratory. Seeds were germinated on MSO medium at 27.degree. C.
with photoperiods of 16 hour light and 8 hour dark Microcarriers
coated with pLD-BD Cry2Aa2 operon DNA was used to bombard sterile
leaves using the Bio-RadPDS-1000/He biolistic device as described
by Daniell (1997). Bombarded leaves were subjected to two rounds of
selection on RMOP medium containing 500 .mu.g/ml of spectinomycin
to regenerate transformants.
[0117] PCR Analysis: DNA was extracted from leaves using the QIAGEN
DNeasy Plant Mini Kit. PCR was done using the Perkin Elmer Gene Amp
PCR System 2400. All PCR reactions were performed using the Qiagen
Taq DNA Polymerase Kit. Primer sequences used were: IP
(5'-ACAATGTAGCCGTACTGGA AGGTGCG GGTG-3'), 1M (5'-CGCGCTT AGC
TGGATAACGCCACGGAA-3), 3P(5'-AAAA CCCGTCCTCA GTTCGGATTGC-3'), and 3M
(5'-CCGCGTTGTITCATCAA GCCTTACG-3'). Samples were run for 30 cycles
with the following sequence: 94.degree. C. for 1 minute, 70.degree.
C. for 1.5 minutes, and 72.degree. C. for 3 minutes. PCR products
were separated on 0.8% agarose gels.
[0118] Southern Blot Analysis: DNA from transformed and
untransformed plants was digested with BgIII and transferred to a
nylon membrane by capillary action. The 0.81 kb probe was generated
by digesting pLD-CtV2 vector DNA with BamHl/BgIII and labeled with
.sup.32P using the ProbeQuant.TM. G-50 Micro Columns (Amersham).
Labeled probe was hybridized with the nylon membrane using the
Stratagene QUICK-HYB hybridization solution and protocol.
[0119] SDS-PAGT Analysis: Transgenic and untransformed leaf
material (600 mg) was ground to a powder in liquid nitrogen.
Protein extraction buffer from the Cry2Aa2 plate kit from
Envirologix (Portland, Me.) used for quantification was added to
the powder and further grinding was performed. The mixture was
centrifuged at 4.degree. C. at 13,000 g for 20 minutes. The
supernatant was removed, boiled in sample buffer, and loaded on a
10% SDS-PAGE gel. The pellet was resuspended in 50 mM NaOH and
centrifuged at 4.degree. C. at 5000 g for 5 minutes to pellet cell
debris. The supernatant was removed, boiled in sample buffer, and
loaded on a 10% SDS-PAGE gel at 200V for 4 hours. Gels were stained
for 2 hours with R-250 Coomassie Blue and destained overnight in
40% methanol and 10% acetic acid. The DC protein assay by Bio-Rad
was used to determine total soluble and pellet protein
concentration as followed by protocol.
[0120] ELISA: A Cry2Aa2 plate kit from Envirologix was used. Leaves
expressing the SG Cry2Aa2, the OD Cry2Aa2, and untransformed
tobacco were quantified and compared. Approximately 20 mg of leaf
was ground in 100 .mu.l of 50 mM NaOH to solubilize CRY proteins.
Transgenic leaf extracts were diluted to fit in the linear range of
the provided Cry2aA2 standard. The .mu.Quant microtiter plate
reader from Bio-Tek read the plate at 450 nanometers (nm). A 1 ppm
Cry2Aa2 standard was supplied by the kit and was used in the line
arrange between 200-1000 ng for quantification. Color development
is proportional to Cry2Aa2 concentration in the sample extract. The
DC protein assay by Bio-Rad was used to determine total soluble
protein concentration as followed by protocol.
[0121] Insect Bioassays: Leaf disc bioassays were conducted on ca.
2 cm.sup.2 excised leaf material, and placed on distilled
water-soaked cardboard lids in 50.times.12 mm plastic petri dishes.
Insects used were susceptible H. virescens (YDK) obtained from Fred
Gould, North Carolina State University, H. zea obtained from the
USDA AIRS facility in Tifton, Ga. and S. exigua from the lab of
William Moar. Insects were tested as five day or ten day old. All
larvae were reared on typical lepidopteran artificial diet prior to
use. Two insects were assayed per leaf sample, except H. zea in
which only 1 insect was added per leaf sample due to the
cannibalistic nature of the insect (2 leaf samples for H. zea). All
leaf samples for each replicate were from the same leaf. Two
samples were evaluated per treatment, and observed daily for
mortality and leaf damage for 5 days. Treatments were replicated
three times.
[0122] Transmission Electron Microscopy and Immunogold Labeling:
Immunogold labeled electron microscopy was performed as described
by A. J. Vrekleij et al.. Sections were first incubated with 0.05M
glycine in PBS buffer (10 mM phosphate buffer, 150 mM NaCl pH 7.4)
for 15 minutes to inactivate residual aldehyde groups. The grids
were then blocked by placing them onto drops of PBS with 5% BSA and
0.1% CWFS gelatin supplemented with 5% normal serum for 30 minutes,
washed on drops of incubation buffer 3 times for 5 min each, and
then incubated for 45 min with the polyclonal Cry2Aa2 to detect
tobacco expression (diluted 1:10,000 in incubation buffer). To
remove unbound primary antibody, sections were washed on drops of
incubation buffer 6.times.5 min each. Sections were then incubated
for 2 hours with a goat anti-rabbit IgG secondary antibody
conjugate to 10 nm gold diluted 1:100 in incubation buffer.
Sections were subsequently washed 6.times.5 minutes in incubation
buffer, 3.times.5 min with PBS, and post-fixed in 2% glutaraldehyde
diluted in PBS for 5 min. Following post-fixation, sections were
washed in PBS 3.times.5 minutes, in distilled water 5.times.2 min
each, and post-stained using uranylacetate and lead citrate.
Sections were then examined in a Zeiss EM 10 transmission electron
microscope at 60 kv.
EXAMPLE 2
[0123] E. Coli Transformants. Due to the similarity of protein
synthetic machinery (Brixey et al. 1997), expression of all metal
resistance conferring chloroplast vectors are first tested in E.
coli before their use in tobacco transformation The activity of the
enzymes, mercury ion reductase (merA) and organomercurial lyase
(merB) are tested by transforming E. coli (XLI-blue) with the
recombinant plasmids and growing them in LB solid medium with
HgCl.sub.2 (FIG. 9). The cells, control (XLI-bue), pLD-merAB and
pLDmerAB-3'UTR are grown in different concentrations of Hg
Cl.sub.2. Control cells do not grow even at concentrations less
than 25 .mu.M Hg Cl.sub.2 but the transformed cells grow well even
at 100 .mu.M HgCl.sub.2-(FIG. 10). The ability to grow at these
high concentrations of mercury in which control is not able to
grow, confirms the functionality of both enzymes. Control and
transformed clones are grown in LB with 500 .mu.g/ml of
spectinomycin for 24 hours at 37.degree. C. When OD.sub.600 is
measured, 1.247 for the clone with 3'UTR, 0.165 for the clone
lacking the 3'UTR, and zero absorbance for the control is observed.
As expected the pLD-merAB-3'UTR transformed clone shows a higher
growth rate probably caused by the 3' effective termination which
allows cells to make more copies of the mer operon transcript that
contain only the aadA, merA and merB genes. In chloroplast genome
we expect a minor effect in the transcription termination
efficiency because the terminator of the genes close to the
cassette and downstream can serve as a terminator, once it is
integrated in the chloroplast genome by homologous
recombination.
[0124] Bombardment and regeneration of chloroplast transgenic
plants: Tobacco (Nicotiana tabacum var. Petit Havana) plants are
grown aseptically by germination of seeds on MSO medium (Daniell
1993). Fully expanded, dark green leaves of about two month old
plants are bombarded as described by Daniell (1997). The plants are
maintained under 500 .mu.g/ml spectinomycin selection in the three
phases; first selection (RMOP medium), second round of selection
(RMOP medium) and third selection MSO (rooting medium) (FIG. 10).
After these selection events, positive transformants are
transferred to soil (FIG. 10). The plants are tested for
integration of the genes in the chloroplast at first round of
selection and before transplanting them to soil. The use of PCR
with specific primers that land in the chloroplast genome and in
the gene cassette allows us to eliminate mutants and show
integration of the selectable marker gene and the mer genes (FIG.
11). After PCR testing, the plants are grown in soil and the seeds
are collected.
[0125] Polymerase Chain Reaction: PCR is done using DNA isolated
from control and transgenic plants to distinguish a) true
chloroplast transformants from mutants and b) chloroplast
transformants from nuclear transformants. Primers for testing the
presence of the aadA (a gene that confers spectinomycin resistance)
in transgenic plants are landed on the aadA coding sequence and 16S
rRNA gene (primers 1IP & 1M). In order to test chloroplast
integration of the mer genes, one primer is landed on the aadA gene
while another is landed on the native chloroplast genome (primers
3P&3M). No PCR product is obtained with nuclear transgenic
plants using this set of primers. The primer set (5P & 2M) is
used to test integration of the entire gene cassette without any
internal deletion or looping out during homologous recombination,
by landing on the respective recombination sites. This screening is
essential to eliminate mutants and nuclear transformants. In order
to conduct PCR analyses in transgenic plants, total DNA from
unbombarded and transgenic plants are isolated as described by
Edwards et al. (1991). Chloroplast transgenic plants containing the
mer gene are moved to second round of selection in order to achieve
homoplasmy.
[0126] Southern Blot Analysis: Southern blots are done to determine
the copy number of the introduced foreign gene per cell as well as
to test homoplasmy. There are several thousand copies of the
chloroplast genome present in each plant cell. Therefore, when
foreign genes are inserted into the chloroplast genome, it is
possible that some of the chloroplast genomes have foreign genes
integrated while others remain as the wild type (heteroplasmy).
Therefore, in order to ensure that only the transformed genome
exists in cells of transgenic plants (homoplasmy), the selection
process is continued. In order to confirm that the wild type genome
does not exist at the end of the selection cycle, total DNA from
transgenic plants is probed with the chloroplast border (flanking)
sequences (the trnI-trnA fragment). If wild type genomes are
present (heteroplasmy), the native fragment size will be observed
along with transformed genomes. Presence of a large fragment (due
to insertion of foreign genes within the flanking sequences) and
absence of the native small fragment should confirm homoplasmy
(Daniell et al., 1998; Kota et al., 1999; Guda et al., 2000).
[0127] The copy number of the integrated gene is determined by
establishing homoplasmy for the transgenic chloroplast genome.
Tobacco Chloroplasts contain 5000-10,000 copies of their genoine
per cell (Daniell et al. 1998). If only a fraction of the genomes
are actually transformed, the copy number, by default, must be less
than 10,000. By establishing that in the transgenics, the merAB
inserted transformed genome is the only one present, one could
establish that the copy number is 5000-10,000 per cell. This is
done by digesting the total DNA with a suitable restriction enzyme
and probing with the flanking sequences that enable homologous
recombination into the chloroplast genome. The native fragment
present in the control should be absent in the transgenics. The
absence of native fragment proves that only the transgenic
chloroplast genome is present in the cell and there is no native,
untransformed, chloroplast genome, without the mer genes present.
This establishes the homoplasmic nature of our transformants,
simultaneously providing us with an estimate of 5000-10,000 copies
of the foreign genes per cell.
[0128] Northern Blot Analysis: Northern blots are done to test the
efficiency of transcription of the merAB operon. Total RNA is
isolated from 150 mg of frozen leaves by using the "Rneasy Plant
Total RNA Isolation Kit" (Qiagen Inc., Chatsworth, Calif.). RNA
(10-40 .mu.g) is denatured by formaldehyde treatment, separated on
a 1.2% agarose gel in the presence of formaldehyde and transferred
to a nitrocellulose membrane (MSI) as described in Sambrook et al.
(1989). Probe DNA (merAB gene coding region) is labeled by the
random-primed method (Promega) with .sup.32P-dCTP isotope. The blot
is pre-hybridized, hybridized and washed as described above for
southern blot analysis. Transcript levels are quantified by the
Molecular Analyst Program using the GS-700 Imaging Densitometer
(Bio-Rad, Hercules, Calif.).
Plant Bloassays
[0129] Germination/Growth Experiments: Seeds of wild-type
(Nicotiana tabacum var Petit Havana), transgenic plant pLD-MerAB,
and transgenic plant pLD-MerAB-3'UTR are sterilized, vernalized at
4' C for at least 24 h, and germinated on 1% Phytoagar plates
(GIBCO/BRL) made with Murashige and Skoog (4.3 g/liter, GIBCO/BRL)
medium containing PMA (phenylmercuric acetate) or mercuric
chloride. Seedlings are grown at 22.degree. C. with a 16 h light/8
h dark period.
[0130] Mercury Vapor Assays: Elemental mercury is relatively
insoluble and volatile and lost quickly from cells and media.
Volatilized Hg.sup.0 is measured on a Jerome 431 mercury vapor
analyzer (Arizona Instrument, Phoeniz, Ariz.) (Rugh, C. L. et al.,
1996). Approximately 5-10 seedlings (10-14 day old, 10-25 mg total
wet weight) are incubated in 2 ml of assay medium (50mm Tris.HCL,
pH 6.8/50 mM NaCl/25 uM HgCl.sub.2) in a 16.times.130 mm test tube
with a side arm for gas removal. The Hg Cl.sub.2 is added to
initiate the assay. The amount of Hg.sup.0 produced is assayed by
bubbling air through the bottom of the sample for 12 sec at 3
cm.sup.3/sec and measuring the release of Hg.sup.0. The time zero
assay will be taken immediately after the seedlings are placed in
the medium. The sample is then reassayed every minute for 10
minutes. The volatilized Hg.sup.0 is measured bypassing the air
sample released from the side arm directly over the gold foil
membrane resistor of a Jerome 431 mercury vapor analyzer. The
instrument is repeatedly standardized with known quantities of
Hg.sup.0 (10-200 ng), reduced from HgCl.sub.2 with excess
SnCl.sub.2. The amount of mercury evolved is normalized by dividing
the number of nanograms of Hg.sup.0 measured by the number of
milligrams of seedling tissue in the assay.
[0131] Photosynthetic studies: From transgenic plants and
untransformed plants, intact chloroplasts are isolated for
photosynthetic studies. O.sub.2 evolution is studied in an oxygen
evolution electrode in the absence or presence of different
concentrations of HgCl.sub.2 and PMA. Electron transport is studied
with suitable electron donors/acceptors to study photosystem I, II
or both. PAGE is used to examine the composition of PSII complex,
especially EP33, after incubation of cells or chloroplasts or
thylakoid membranes with different concentrations of HgCl.sub.2 and
PMA. In vivo chloroplast fluorescence is studied to monitor changes
in control and transformed cells or chloroplasts to measure Fo, Fm,
Fv.
[0132] Inheritance of Introduced Foreign Genes: While it is
unlikely that introduced DNA would move from the chloroplast genome
to nuclear genome, it is possible that the gene could get
integrated in the nuclear genome during bombardment and remain
undetected in Southern analysis. Therefore, in initial tobacco
transformants, some are allowed to self-pollinate, whereas others
are used in reciprocal crosses with control tobacco (transgenics as
female accepters and pollen donors; testing for maternal
inheritance). Harvested seeds (T1) are germinated on media
containing spectinomycin. Achievement of homoplasmy and mode of
inheritance is classified by looking at germination results.
Homoplasmy is indicated by totally green seedlings (Daniell et al.,
1998) while heteroplasmy is displayed by variegated leaves (lack of
pigmentation, Svab & -Maliga, 1993). Lack of variation in
chlorophyll pigmentation among progeny also underscores the absence
of position effect, an artifact of nuclear transformation. Maternal
inheritance is demonstrated by sole transmission of introduced
genes via seed generated on transgenic plants, regardless of pollen
source (green seedlings on selective media). When transgenic pollen
is used for pollination of control plants, resultant progeny would
not contain resistance to chemical in selective media (will appear
bleached; Svab and Maliga, 1993). Molecular analyses confirms
transmission and expression of introduced genes, and T2 seed are
generated from those confirmed plants by the analyses described
above.
EXAMPLE 3
[0133] Chlorella vulgaris transformation vector: The region 16S to
23S of the Chlorella vulgaris chloroplast genome is amplified by
PCR using specific primers complementary to rrn16 and to rrn23. The
PCR product will be cloned into pCR 2.1 vector available from
Promega. The PCR product 16S to 23S is removed from the pCR2.1
vector by a blunt end restriction endonuclease and cloned into the
pUC19 in which the multiple cloning site has been removed using a
blunt end restriction enzyme (Pvu1I). Then the cassette containing
the promoter, the antibiotic resistance gene and the merAB genes is
inserted into the new vector (Chlorella transformation vector)
using a blunt end restriction enzyme (HincII) that is present in
the spacer region between trnA and trnT. The final construct is
used for the transformation of Chlorella vulgaris (FIG. 12).
[0134] Bombardment and transformation of Chlorella vulgaris: The
biolistic transformation method (Sanford et al. 1993) is optimized
for transformation of Chlorella vulgarism. Chlorella is grown in
liquid heterotrophic medium (5 sporulation agar) at 25.degree. C.
to late-log phase (.about.6.times.10.sup.6 cells/ml). To prepare a
monolayer for bombardment (2.times.10.sup.7), cells are collected
onto prewetted 45 mm GVWP filters (Millipore) under gentle (30
mBar) vacuum. Gold particles are coated with the transforming
plasmid. (Sanford et al. 1993) The monolayer filters are bombarded.
Immediately after bombardment, filters are transferred to selective
solid media containing 500 .mu.g/ml spectinomycin and incubated at
22.degree. C. under high light. After approximately 6 weeks, green
colonies are picked from a background of bleached cells onto
selective plates and grown for an additional 1-2 weeks. Colonies
are harvested and screened for integration of foreign genes when
they reached a diameter of approximately 5 mm.
[0135] Chlorella vulgaris Bioassays: Growth and colonies formation
bioassay are performed as explained in the plant germination growth
experiment; the only change is the use of Chlorella specific
media.
[0136] Mercury vapor assays: Mercury vapor assays are performed in
the way explained for plants above except changing the growth media
specific to Chlorella, including temperature and light
intensity.
[0137] Photosynthetic studies: Photosynthetic studies are performed
in the way explained for plants above except untransformed and
transformed Chlorella cells will be directly used to study the
effect of mercury toxicity.
EXAMPLE 4
[0138] Synechocystis transformation vector: The region 16S to 23S
of the Synechocystis genome is amplified by PCR using specific
primers complementary to rrn 116 and to rrn23. The PCR product is
cloned into the pCR 2.1 vector available from Promega. The PCR
product 16S to 23S is removed from the pCR2.1 vector by a blunt end
restriction endonuclease and cloned into pUC19 in which the
multiple cloning site has been removed using a blunt end
restriction enzyme (Pvu1I). Then the cassette containing the
promoter, the antibiotic resistance gene and the merAB genes is
inserted into the new vector (Synechocystis transformation vector)
using a blunt end restriction enzyme (HincII) that is present in
the spacer region between trnI and trnA. The final construct will
be used for the transformation of Synechocystis (FIG. 13).
[0139] Transformation of Synechocystis: A fresh culture of wild
type in BG-11 (heterotrophic medium) plus glucose is grown to
OD.sub.730=0.5 after 2-3 days of culture. Cells are spun down in
sterile 50 ml tubes at room temperature and resuspended in the
original growth medium to OD.sub.730=2.5. Transforming DNA is added
to resuspended cells in sterile glass culture tubes. Tubes are
placed in rack in the growth chamber at 30.degree. C. for 6 hours
and shaken for 3 hours. Cells (200 .mu.l) are plated on a sterile
filter that has been placed on a BG-11 plus glucose plate and
spread around. After growth for 24 hours and they are transferred
to filters on appropriate medium containing spectinomycin or
mercuric chloride.
[0140] Synechocystis Bioassays: Growth and colonies formation
bioassay are performed as explained in the plant germination-growth
experiment; the only change is the use Synechocystis growth
media.
[0141] Mercury vapor assays: Mercury vapor assays are performed in
the way explained for plants above except changing the growth media
specific to the Synechocystis, including temperature and light
intensity.
[0142] Photosynthetic studies: Photosynthetic studies are performed
in the way explained for plants above except untransformed and
transformed Synechocystis cells are directly used to study the
effect of mercury toxicity.
EXAMPLE 5
[0143] Lemna transformation vector: The Lemna chloroplast vector,
as shown in FIG. 14, is constructed in the same way as explained
above for tobacco, with the exception that the Lemna chloroplast
DNA flanking sequences are used.
[0144] Bombardment and regeneration of transgenic plants: Lemna
plants are transformed and regenerated in the way explained for
tobacco in Example 1 above.
[0145] Plant Bioassays: Various plant bioassays are performed as
explained for tobacco in Example 1 above.
EXAMPLE 6
[0146] Sugarcane transformation vector: The Sugarcane chloroplast
vector, as shown in FIG. 15, is constructed in the same way as
explained above for tobacco, with the exception that the Sugarcane
chloroplast DNA flanking sequences are used.
[0147] Bombardment and regeneration of transgenic plants: Sugarcane
plants are transformed and regenerated in the way explained for
tobacco in Example 1 above.
[0148] Plant Bioassays: Various plant bioassays are performed as
explained for tobacco in Example 1 above.
References
[0149] Begley T P A, Walts A E, Walsh Conn. (1986) Mechanistic
studies of a protonolytic organomercurial cleaving enzyme:
bacterial organomercurial lyase. Biochemistry 25: 7192-7200.
[0150] Bernier M, Popovic R, Carpentier R (1993) Mercury inhibition
of photosystem 11. FBS Lett. 32:19-23.
[0151] Bernier M, Carpentier R (1995) The action of mercury on the
binding of extrinsic polypeptides associated with water oxidizing
complex of photosystem II.
[0152] Bizily S, Rugh C C, Summers A O, Meagher R B (1999)
Phytoremediation of methyl mercury pollution: merB expression in
Arabidopsis thaliana plants confer resistance to organomercurial.
PNAS 96: 6808-6813.
[0153] Bizily S, Rugh C C, Meagher R B (2000) Phytoremediation of
hazardous organomercurials by genetically engineered plants. Nature
Biotechnology 18; 213-217.
[0154] Bogorad, L. Engineering chloroplasts: an alternative site
for foreign genes, proteins, reactions, and products. Trends in
Biotechnology. 18, 257-263 (2000).
[0155] Bradley, D. et al. The insecticidal CrylB crystal protein of
Bacillus thuringiensis ssp. Thuringiensis has dual specificity to
coleopteran and lepidopteran larvae. J. Invert. Pathol. 65, 162-173
(1995).
[0156] Carlson P S (1973) The use of protoplasts for genetic
research. Proc. Natl. Acad. Sci. USA 70:.598-602.
[0157] Compeau G C, Bartha R (1985) Sulfate-reducing bacteria:
principal methylators in freshwater sediments. Appl. Environ.
Microbiol, 50: 498-502.
[0158] Crickmore, N. & Ellar, D. Involvement of a possible
chaperonin in the efficient expression of a cloned crylIA
8-enclotoxin gene in Bacillus thuringiensis. MoL Microbiol. 6,
1533-1537 (1992).
[0159] Crickmore, N., Wheeler, V. & Ellar, D. Use of an operon
fusion to induce expression and crystallisation of a Bacillus
thuringiensis 5-endotoxin encoded by a cryptic gene. MoL Gen. Genet
242, 365-368 (1994).
[0160] Daniell, H. Foreign gene expression in chloroplasts of
higher plants mediated by tungsten particle bombardment. Methods
Enzymo/. 217, 536-556 (1993).
[0161] Daniell, H. et al. Engineering plants for stress tolerance
via organelle genomes. NATO ASI Series. 86, 589-592 (1994).
[0162] Daniell, H. Transformation and foreign gene expression in
plants mediated by microprojectile bombardment. Meth. Mol. Biol.
62,463-489(1997).
[0163] 1 A operons on the formation of Cry2A inclusions in Bacillus
thuringiensis. FEMS Microbiol. Lett. 165, 35-41 (1998).
[0164] Daniell, H. et al. Containment of a herbicide resistance
through genetic engineering of the chloroplast genome. Nature
Biotechnol. 16, 345-348 (1998).
[0165] Daniell, H. GM crops: public perception and scientific
solutions. Trends in Plant Science. 4, 467-469 (1999).
[0166] Daniell, H. New tools for chloroplast genetic engineering.
Nat. Biotechno'll. 17, 855-856 (1999).
[0167] Daniell H, Kulandaivelu G and Chandrasingh U (1980)
Substituted p-benzoquinones having high electron affinity as
photosystem II electron acceptors. Z. Naturforsch. 35C,
136-138.
[0168] Daniell H and Sarojini G (1981) Site of action of
2,5-dimethoxy-3,6-dichloro-p-benzoqliinone in the photosynthetic
electron transport chain. Z. Naturforsch. 36C, 656-661.
[0169] Daniell H, Rarnanujan P, Krishnan M, Gnanam A, Rebeiz C A
(1983) In vitro synthesis of photosynthetic membranes: L
Development of photosystem I activity and cyclic phosphorylation.
Biochem. Biophys. Res. Comun. 11 1: 740-749.
[0170] Daniell H, Krishnan M, Renganathan M and Gnanam A (1984)
Radioisotopic evidence for the polypeptides associated with
photosystem II activity. Biochem. Biophys. Res. Commun.
125,988-995.
[0171] Daniell H, Anbudurai P R, Periyanan S, Renganathan M,
Bhardwaj R, Kulandaivelu G and Gnanam A(1985) Oxygenic
photoreduction of methyl viologen without the involvement of
photosystem I during plastid development. Biochem. Biophys. Res.
Commun. 126, 1114-1121.
[0172] Daniell H and McFadden B A (1986) Characterization of DNA
uptake by the cyanobacterium Anacystis nidulans. Mol. Gen. Genetics
204, 243-248.
[0173] Daniell H, Sarojini G and McFadden B A (1986) Transformation
of the cyanobacterium Anacystis nidulans 6301 with the Escherichia
coli plasmid pBR322. Proc. Natl. Acad. Sci. USA 83, 2546-2550.
[0174] Daniell H and McFadden B A (1987) Uptake and expression of
bacterial and cyanobacterial genes by isolated cucumber etioplasts.
Proc. Natl. Acad. Sci. USA 84: 6349-6353. Daniell H and McFadden B
A (1988) Genetic Engineering of plant chloroplasts. U.S. Pat. Nos.
5,932,479; 5,693,507.
[0175] Daniell H, Vivekananda J, Neilsen B, Ye G N, Tewari K K,
Sanford J C (1990) Transient foreign gene expression in
chloroplasts of cultured tobacco cells following biolistic delivery
of chloroplast vectors. Proc. Natl. Acad. Sci. USA 87: 8892.
[0176] Daniell H, Krishnan M, McFadden B A (1991) Expression of
B-glucuronidase gene in different cellular compartments following
biolistic delivery of foreign DNA in to wheat leaves and calli.
Plant Cell Reports 9: 615-619.
[0177] Daniell H, Muthukumar B and Lee S B (2001) Marker free
transgenic plants: engineering the chloroplast genome without the
use of antibiotic selection. Current Genetics. In press.
[0178] DeCosa B, Moar W, Lee S B, Miller M, Daniell H (2001).
Hyper-expression of the Bt Cry2Aa2 operon in chloroplasts leads to
formation of insecticidal crystals. Nature Biotechnol. 19:
71-74.
[0179] De Wilte, C. et al. (2000). Plants as Bioreacters for
Protein Production: Avoiding the Problem of Transgene Silencing.
Plant Molecular Biology 43:347-389.
[0180] Edwards K, Johnstone C, Thompson C (1991) A simple and rapid
method for preparation of plant genomic DNA for PCR analysis.
Nucleic Acids Res. 19: 1349.
[0181] Foster T J (1983) Plasmid-determined resistance to
antimicrobial drugs and toxic metal ions in bacteria. Microbiol.
Rev. 47: 361-409.
[0182] Ge, B. et al. Differential effects of helper proteins
encoded by the cry2A and cryl Greenplate, J. Quantification of
Bacillus thuringiensis insect control protein CrylAc over time in
bollgard cotton fruit and terminals. J. Econ. Entomo/. 92,
1377-1383 (1999).
[0183] Gilmour C C, Henry E A, Mitchel R (1992) Sulfate stimulation
of mercury methylation in freshwater sediments. Environ. Sci.
Technol. 26: 2281-2287.
[0184] Guda, C., Lee, S. B. & Daniell, H. Stable expression of
a biodegradable protein-based polymer in tobacco chloroplasts.
Plant Cell Reports. 19, 257-262 (2000).
[0185] Harada M, Minarnata Disease Research Group (1995) Minamata
disease: methylmercury poisoning in Japan caused by environmental
pollution. Crit. Rev. Toxicol. 25: 1-24.
[0186] Kota, et al. Overexpression of the Bacillus thurin iensis
(13t) Cry2Aa? protein 9 in chloroplasts confers resistance to
plants against susceptible and Bt-resistant insects. Proc. Nati.
Acad. Sci. 96,1840-1845 (1999).
[0187] Kulandaivelu G and Daniell H. (1980) Dichlorophenyl
dimethylurea (DCMU) induced increase in chlorophyll a fluorescence
intensity: An index of photosynthetic oxygen evolution in leaves,
chloroplasts and algae. Physiol. Plant. 48, 385-388.
[0188] Ma, J. et al. Generation and assembly of secretary
antibodies in plants. Science. 268, 716-719 (1995).
[0189] McBride K E, Svab Z, Schaaf D J, Hogen P S, Stalker D M,
Maliga P (1995) Amplification of a chimeric Bacillus gene in
chloroplasts leads to extraordinary level of an insecticidal
protein in tobacco. Bio/technology 13; 362-365.
[0190] Minamata Disiase Research Group (1968)Minamata Disease.
(Medical School of Kumamoto University, Kumamoto, Japan)
[0191] Moar, W. et al. Insecticidal activity of the CryllA protein
from the NRD-12 isolate of Bacillus thuringiensis subsp. kurstaki
expressed in Escherichia coli and Bacillus thuringiensis and in a
leaf-colonizing strain of Bacillus cereus. Appl. Environ.
Microbiol. 60, 896-902 (1994).
[0192] Moar, W. et al. Development of Bacillus thuringiensis Cryl C
resistance by Spodoptera exigua. Appl. Environ. Microbiol. 61,
2086-2092 (1995).
[0193] Navrath, C., Poirier, Y., & Somerville, C. Targeting of
the polyhydroxybutyrate biosynthetic pathway to the plastis of
Arabidopsis thaliana results in high levels of polymer
accumulation. Proc. Natl. Acad. Sci. 91, 12760-12764 (1994).
[0194] Peerenboom, E (2000) German health minister calls time out
for Bt maize. Nature Biotechnol. 18: 374.
[0195] Puchta H (2000) Removing selectable marker genes: taking the
shortcut. Trends in plant Science 5:273-274.
[0196] Rashid A, Popovic R (1990) Protective role of CaCl.sub.2
against Pb.sup.2+ inhibition in photosystem IL FEBS Lett. 271:
181-184.
[0197] Roy, H. & Nierzwicki-Bauer, S. RuBisCo: genes,
structure, assembly and evolution. The Photosynthetic Apparatus, L.
Bogorad, 1. Vasil (eds),pp 347-364, Academic Press, NY. (1994).
[0198] Rugh C C, Summers A O, Meagher R B (1996) Mercuric ion
reductase and resistance in transgenic Arabidopsis thaliana plants
expressing modified bacterial merA gene. PNAS 93: 3182-3187.
[0199] Salt D E et al. (1998) Phytoremediation. Rev. Plant.
Physiol. Plant. Mol. Biol. 49-643-668.
[0200] Sanford J C, Smith F D, Russell J A (1993) Optimizing the
biolistic process for different biological applications. Methods
Enzymol. 217: 483-509.
[0201] Sambrook J, Fritch E F, Maniatis T (1989) Molecular cloning.
Cold Spring Harbor Press, Cold Spring Harbor, N.Y.
[0202] Sarojini G, Daniell H and Vermaas W F J (1981) Site of
electron acceptance by 3,6,dichloro-2,5-dimethoxy-p-benzoquinone
and its relation to the bicarbonate effect on photosynthetic
electron transport Biochem. Biophys. Res. Commun. 102, 944-951.
[0203] Sidorov V A, Kasten D, Pang S Z, Hajdukiewicz P T J, Staub J
M, Nehra N S (1999) Stable chloroplast transformation in potato:
use of green fluorescent protein as a plastid marker. Plant Journal
19:209-216.
[0204] Staub J M, Garcia B, Graves J, Hajdukiewicz P T J, Hunter P,
Nehra N, Paradkar V, Schlittler M, Caroll J A, Spatola I, Ward D,
Ye G, Russell D (2000) High-yield production of human therapeutic
protein in tobacco chloroplast. Nature Biotechnol. 18: 333-338.
[0205] Summers A O, Silver S (1978) Microbial transformation of
metals. Annu. Rev. Microbiol. 32:637-672.
[0206] Summers A O (1986) Organization, expression, and evolution
of genes for mercury resistance. Annu. Rev. Microbiol 40:
607-634.
[0207] Svab Z, Maliga P (1993) High frequency plastid
transformation in tobacco by selection for a chimeric aadA gene.
Proc. Natl. Acad. Sci. USA 90: 913-917.
[0208] Trebs A (1980) Inhibitors in electron flow: tools for the
functional and structural localization of carriers and energy
conservation sites.
[0209] A. J Vrekleij, J. M. Leunissen (eds.), Immuno-gold Labeling
in Cell Biology CRC Press, Boca Raton, Fla. (1989).
[0210] Yamamoto, T. & lizka, T. Two types of entomocidal toxins
in the parasporal crystals of Bacillus thuringiensis var. kurstaki.
Archives Biochem. Biophys. 227, 233241 (1983).
[0211] Ye, X et al, Engineering the provitamin A (0-carotene)
bipsynthetic pathway into (carotenoid-free) rice enclosperm.
Science. 287, 303-305 (2000).
[0212] Herbicide Resistance Crops, Agricultural, Environmental,
Economic, Regulatory and Technical Aspects, Duke, S. O., edt., CRC
Press, Inc. (1996).
[0213] Herbicide Resistance in Plants, Biology and Biochemistry,
Powles, S. B., and Holtum, J A M., eds., CRC Press, Inc.
(1994).
[0214] Peptides: Design, Synthesis, and Biological Activity,
Basava, C and Anantharmaiah, G. M., eds., Birkhauser Boston,
1994.
[0215] Protein Folding: Deciphering the Second Half of the Genetic
Code, Gierasch, L. M., and King, J., eds., American Association For
the Advancement of Science (1990).
Sequence CWU 1
1
6 1 30 DNA Artificial Sequence Description of Artificial Sequence
Primer 1 acaatgtagc cgtactggaa ggtgcgggtg 30 2 27 DNA Artificial
Sequence Description of Artificial Sequence Primer 2 cgcgcttagc
tggataacgc cacggaa 27 3 25 DNA Artificial Sequence Description of
Artificial Sequence Primer 3 aaaacccgtc ctcagttcgg attgc 25 4 25
DNA Artificial Sequence Description of Artificial Sequence Primer 4
ccgcgttgtt tcatcaagcc ttacg 25 5 24 DNA Artificial Sequence
Description of Artificial Sequence Primer 5 ctgtagaagt caccattgtt
gtgc 24 6 25 DNA Artificial Sequence Description of Artificial
Sequence Primer 6 tgactgccca acctgagagc ggaca 25
* * * * *
References